Immunogenic peptides specific to bcma and taci antigens for treatment of cancer

ABSTRACT

This disclosure relates to immunogenic peptides that are specific to B-cell maturation antigen (BCMA) and Transmembrane activator and CAML interactor (TACI), and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Division of U.S. patent application Ser. No.16/641,722, filed Feb. 25, 2020, which is a U.S. National Stageapplication of International Application No. PCT/US2018/049260, filedAug. 31, 2018, which claims the benefit of U.S. Provisional ApplicationNo. 62/553,669, filed on Sep. 1, 2017. The entire contents of each ofthe foregoing applications are incorporated herein by reference.

STATEMENT OF FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos. P50CA100707, P0l CA078378, and R0l CA050947 awarded by the NationalInstitutes of Health. The Government has certain rights in theinvention.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submittedelectronically as an XML file named 00530-0337002_SL_ST26.xml. The XMLfile, created on Oct. 28, 2022, is 31,686 bytes in size. The material inthe XML file is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

This disclosure relates to immunogenic peptides that are specific toB-cell maturation antigen (BCMA) and Transmembrane activator and CAMLinteractor (TACI), and methods of use thereof.

BACKGROUND

Cancer is currently one of the diseases that have the highest humanmortality. According to the World Health Organization statistical data,in 2012 the number of global cancer incidence and death cases reached 14million and 8.2 million, respectively. In the United States, cancer isresponsible for at least 25% of all deaths.

In recent years, new therapies have been developed for treating varioustypes of cancers. Patients afflicted with cancers are often treated byusing, e.g., surgeries, chemotherapies and/or immune therapies. Theprognosis for these patients sometimes is still unsatisfactory.Efficacious therapies and/or prophylactic regimens for treating thecancer are therefore urgently needed.

SUMMARY

This disclosure relates, in part to, to immunogenic peptides, T cells(e.g., CD8⁺ cytotoxic T cells (CTL) and/or CD4⁺ helper T cells), andnanoparticles (e.g., polymeric nanocarriers or liposomal nanoparticles)encapsulating peptides that are specific to B-cell maturation antigen(BCMA) or Transmembrane activator and CAML interactor (TACI), andmethods of use thereof.

In one aspect, the disclosure relates to a peptide comprising,consisting essentially of, or consisting of, an amino acid sequence thatis identical to the amino acid sequence set forth in any one of SEQ IDNO: 13-17, or differs by 1 to 6 amino acid residues. In someembodiments, the amino acid at position 1 of SEQ ID NOs: 13-17 isunaltered. In some embodiments, the amino acid(s) at one or more ofpositions 1, 2, or 9 of SEQ ID NOs: 13-17 is substituted with anotheramino acid. For example, position 1, 2, or 9 is substituted; positions 1and 2 are substituted; positions 2 and 9 are substituted; positions 1and 9 are substituted; or positions 1, 2, and 9 are substituted. Incertain embodiments, the peptide is 9 to 30 amino acids in length (i.e.,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30).

In some embodiments, the amino acid sequence is SEQ ID NO: 13, SEQ IDNO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

In some embodiments, the peptide comprises an amino acid sequence setforth in any one of SEQ ID NO: 13-17 wherein the peptide includes 1 to15 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acidsat the N- and/or C-terminus of the amino acid sequence.

In another aspect, the disclosure relates to a peptide comprising,consisting essential of, or consisting of, a first amino acid sequenceconsisting of an amino acid sequence that is at least 40%, at least 45%,at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, or at least 95%identical to any one of SEQ ID NOs: 1-17; and a second amino acidsequence that is heterologous to the first amino acid sequence. In someembodiments, the amino acid(s) at one or more of positions 1, 2, or 9 ofSEQ ID NOs: 1-17 is substituted with another amino acid. For example,position 1, 2, or 9 is substituted; positions 1 and 2 are substituted;positions 2 and 9 are substituted; positions 1 and 9 are substituted; orpositions 1, 2, and 9 are substituted. In certain embodiments, thepeptide is 9 to 30 amino acids in length (i.e., 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30).

In another aspect, the disclosure relates to a peptide comprising,consisting essentially of, or consisting of an amino acid sequence thatis at least 40%, at least 45%, at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, or at least 95% identical to any one of SEQ ID NOs: 1-17. Insome embodiments, the amino acid(s) at one or more of positions 3, 4, 5,6, 7, or 8 of SEQ ID NOs: 1-17 is substituted with another amino acid.For example, position 3, 4, 5, 6, 7, or 8 is substituted; positions 3and 4 are substituted; positions 3 and 5 are substituted; positions 3and 6 are substituted; positions 3 and 7 are substituted; positions 3and 8 are substituted; positions 4 and 5 are substituted; positions 4and 6 are substituted; positions 4 and 7 are substituted; positions 4and 8 are substituted; positions 5 and 6 are substituted; positions 5and 7 are substituted; positions 5 and 8 are substituted; positions 6and 7 are substituted; positions 6 and 8 are substituted; positions 7and 8 are substituted; or any combination of three different, 4different, 5 different, or 6 different positions from the group ofpositions 3, 4, 5, 6, 7, and 8 are substituted.

In some embodiments, the peptide binds to a major histocompatibilitycomplex (WIC) molecule. In some embodiments, wherein the peptide, inassociation with a MHC molecule, is recognized by an antigen specific Tcell receptor on a T cell. In some embodiments, the MHC molecule is anMHC class I molecule or an MHC class II molecule. In some embodiments,the MHC class I molecule is HLA-A (e.g., HLA-A2, HLA-A24, HLA-A1,HLA-A3, HLA-A30, HLA-A26, HLA-A68, or HLA-A11), HLA-B or HLA-C. In someembodiments, the MHC molecule is an HLA-A2 molecule or a HLA-A24molecule.

The disclosure also provides a composition comprising the peptide asdescribed herein and a second agent. In some embodiments, the secondagent is selected from the group consisting of compounds to enhance theBCMA and TACI-specific responses such as (1) Cytokines and Chemokines;(2) checkpoint inhibitors including anti-PD1, anti-PDL1, anti-CTLA4,anti-LAG3, and anti-TIM3; (2) immune agonists including anti-CD28,anti-CD40L (CD154), anti-41BB (CD137), anti-OX40 and anti-GITR, (3)immune modulators including lenalidomide, pomalidomide, a Thalidomideanalogue, IMiDS compound, and/or HDAC inhibitors (e.g., ACY241) as asingle agent and/or in combination with Dexamethasone; (4) adjuvant; (5)therapeutics which increase the BCMA and TACI-specific responsesincluding with vaccine, cell therapies and/or antibodies; (6)therapeutics which alternate the BCMA and TACI-specific responsesincluding peptide-based vaccine, different types of vaccine (RNAvaccine, DNA vaccine), cell therapies, specific modulators and/orspecific inhibitors; and (7) therapeutics that have an independentapproach from the BCMA and TACI-targeting therapy to widely cover immuneresponses to the disease including biological and non-biologicalapproaches. In some embodiments, the second agent is an immunestimulatory agent (e.g., a cytokine or a T helper epitope). In someembodiments, the second agent is a T helper epitope. In someembodiments, the T helper epitope is a PADRE sequence or a universalTetanus Toxoid T helper (TT Th) epitope. In some embodiments, the secondagent is an adjuvant. The adjuvant can be selected from the groupconsisting of Freund's complete adjuvant, Freund's incomplete adjuvant,alum, a ligand for a Toll receptor, QS21, MI, cholera toxin (CT), E.coli heat labile toxin (LT), mutant CT (MCT), and mutant E. coli heatlabile toxin (MLT). In some embodiments, the second agent is a toll likereceptor-3 ligand (e.g., Poly ICLC), interferon alpha (IFNα), interferongamma (IFNγ), Granulocyte-macrophage colony-stimulating factor (GM-CSF),anti-interleukin 6 (IL-6), IL-6 inhibitor, an anti-OX40 antibody, ananti-GITR antibody. In some embodiments, the second agent is acheckpoint inhibitor (e.g., anti-LAG3 antibody). In some embodiments,the second agent is an immune modulator including lenalidomide,pomalidomide, a Thalidomide analogue, IMiDS compound, and/or HDACinhibitors (e.g., ACY241) as a single agent and/or in combination withDexamethasone.

In one aspect, the disclosure also relates to a pharmaceuticalcomposition comprising the peptide as described herein and apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical composition comprises an agent selected from the groupconsisting of compounds to enhance the BCMA and TACI-specific responsessuch as (1) Cytokines and Chemokines; (2) checkpoint inhibitorsincluding anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, and anti-TIM3; (2)immune agonists including anti-CD28, anti-CD40L (CD154), anti-41BB(CD137), anti-OX40 and anti-GITR, (3) immune modulators includinglenalidomide, pomalidomide, a Thalidomide analogue, IMiDS compound,and/or HDAC inhibitors (e.g., ACY241) as a single agent and/or incombination with Dexamethasone; (4) adjuvant; (5) therapeutics whichincrease the BCMA and TACI-specific responses including with vaccine,cell therapies and/or antibodies; and (6) therapeutics that have anindependent approach from the BCMA and TACI-targeting therapy to widelycover immune responses to the disease. In some instances, thepharmaceutical composition includes one or more of: an adjuvant (e.g.,Freund's complete adjuvant, Freund's incomplete adjuvant, alum, a ligandfor a Toll receptor, QS21, RIBI, cholera toxin (CT), E. coli heat labiletoxin (LT), mutant CT (MCT), and mutant E. coli heat labile toxin(MLT)); an immune agonist (e.g., an anti-OX40 antibody, an anti-GITRantibody); a checkpoint inhibitor (e.g., anti-LAG3 antibody); or animmune modulator (e.g., lenalidomide, pomalidomide, a Thalidomideanalogue, IMiDS compound, and/or HDAC inhibitors (e.g., ACY241) as asingle agent and/or in combination with Dexamethasone).

In some embodiments, the pharmaceutical composition further comprises acheckpoint inhibitor. In one embodiment, the checkpoint inhibitor is ananti-LAG3 antibody. In some embodiments, the pharmaceutical compositionfurther comprises lenalidomide. In some embodiments, the pharmaceuticalcomposition further comprises lenalidomide, pomalidomide, a Thalidomideanalogue, IMiDS compound and/or HDAC inhibitors (e.g., ACY241) as asingle agent and/or in combination with Dexamethasone.

In some embodiments, the pharmaceutical composition further comprises aT cell (e.g., a CTL) specific for BCMA. In some embodiments, thepharmaceutical composition further comprises a T cell (e.g., a CTL)specific for TACI. In certain instances, the CTL is a CTL obtained byexposure to a peptide comprising or consisting of one or more of SEQ IDNO: 13 or 14. In other instances, the CTL is a CTL obtained by exposureto a peptide comprising or consisting of any one or more of SEQ ID NOs:15-17. In some instances, the CTL is a memory CD8⁺ CTL. In someinstances, the CTL is a memory CD8⁺ CD45RO⁺ CTL. In some instances, theCTL is a non-memory CD8⁺ CTL. In some instances, the CTL is an effectorCD8⁺ CTL. In some instances, the CTL is activated CD8⁺ CTL. In someinstances, the CTL is a Tetramer-positive CD8⁺ CTL. In some instances,the CTL is a CD8⁺ CTL that has upregulated a costimulatory moleculeexpression. In some instances, the CTL is a CD8⁺ CTL that hasupregulated a checkpoint molecule expression. In some instances, the CTLis a CD8⁺ CTL that produce cytokine(s) and/or that has upregulatedcritical cytolytic marker(s) (e.g. CD107, Granzyme, Perforin) expressionand/or production. In some instances, the CTL is a CD8⁺ CTL that hasactivities against tumor or other targets.

In one aspect, the disclosure relates to a nucleic acid encoding apeptide as described herein. In certain instances, the nucleic acid is aRNA (e.g., mRNA). In other embodiments, the nucleic acid is a DNA. Insome instances, the RNA or DNA is encapsulated in a nanocarrier (e.g.,polymeric such as PLGA or liposomal). The RNA and DNA may comprisesother regulatory sequences (e.g., start codon, stop codon, polyA tail).

In one aspect, the disclosure also relates to a vector comprising anucleic acid encoding the peptide as described herein. In someembodiments, the nucleic acid sequence is operably linked to a promoter,a regulatory element, or an expression control sequence.

In another aspect, the disclosure also relates to a cultured cellcomprising the vector as described herein. In some embodiments, the cellis a mammalian cell, a human cell, or an immune cell.

In another aspect, the disclosure provides a virus comprising a nucleicacid encoding the peptide as described herein. In some embodiments, thevirus is a lentivirus, an adenovirus, an adeno-associated virus, a humanfoamy virus, parvovirus, myxoma virus, Newcastle disease virus, areovirus, Seneca valley virus, measles virus, poliovirus, vacciniavirus, herpes simplex virus, or vesicular stomatitis virus.

In one aspect, the disclosure relates to a combination of at least twodifferent peptides, wherein the at least two different peptides areselected from the group of peptides having an amino acid sequence setforth in SEQ ID NOs: 13-17. The combination can include peptides with 1to 4 substitutions in one or more of SEQ ID NOs: 13-17. In someinstances, the substitutions are at one or more of positions 1, 2, or 9.In some instances, position 1 is not altered. In some instances, thepeptides are 9 to 30 amino acids in length. In some embodiments, thecombination comprises at least 2, 3, 4, or all 5 peptides having anamino acid sequence set forth in SEQ ID NOs: 13-17. In some instances,the combination comprises two or more peptides set forth in SEQ ID NOs:13-17, wherein the two or more peptides have 1 to 15 (i.e., 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids at the N- and/orC-terminus of the amino acid sequence.

In one aspect, the disclosure also relates to a pharmaceuticalcomposition comprising the combination of peptides as described herein;and a pharmaceutically acceptable carrier. In some embodiments, thepharmaceutical composition comprises an agent selected from the groupconsisting of compounds to enhance the BCMA and TACT-specific responsessuch as (1) Cytokines and Chemokines; (2) checkpoint inhibitorsincluding anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, and anti-TIM3; (2)immune agonists including anti-OX40 and anti-GITR, (3) immune modulatorsincluding lenalidomide, pomalidomide, a Thalidomide analogue, IMiDScompound, and/or HDAC inhibitors (e.g., ACY241) as a single agent and/orin combination with Dexamethasone; (4) adjuvant; (5) therapeutics whichincrease the BCMA and TACI-specific responses including with vaccine,cell therapies and/or antibodies; and (6) therapeutics that have anindependent approach from the BCMA and TACI-targeting therapy to widelycover immune responses to the disease. In some instances, thepharmaceutical composition includes one or more of: an adjuvant (e.g.,Freund's complete adjuvant, Freund's incomplete adjuvant, alum, a ligandfor a Toll receptor, QS21, RIBI, cholera toxin (CT), E. coli heat labiletoxin (LT), mutant CT (MCT), and mutant E. coli heat labile toxin(MLT)); an immune agonist (e.g., an anti-OX40 antibody, an anti-GITRantibody); a checkpoint inhibitor (e.g., anti-LAG3 antibody); and/orlenalidomide, pomalidomide, a Thalidomide analogue, IMiDS compound,and/or HDAC inhibitors (e.g., ACY241) as a single agent and/or incombination with Dexamethasone.

In some embodiments, the pharmaceutical composition further comprises animmune agonist. In some instances, the immune agonist can be ananti-OX40 antibody or an anti-GITR antibody. In some embodiments, thepharmaceutical composition further comprises a checkpoint inhibitor. Inone instance, the checkpoint inhibitor is an anti-LAG3 antibody. In someembodiments, the pharmaceutical composition further compriseslenalidomide, pomalidomide, a Thalidomide analogue, IMiDS compound,and/or HDAC inhibitors (e.g., ACY241) as a single agent and/or incombination with Dexamethasone. In one aspect, the disclosure alsoprovides a composition comprising an isolated dendritic cell, whereinthe dendritic cell presents a peptide sequence on its surface, whereinthe peptide sequence comprises at least one major histocompatibilitycomplex (MEW) class I peptide epitope of one or both of BCMA antigen(SEQ ID NO: 18) and TACI antigen (SEQ ID NO: 19).

In some embodiments, the MEW class I peptide epitope is an HLA-A2peptide epitope.

In some embodiments, the MEW class I peptide epitope is an HLA-A24peptide epitope.

In some embodiments, the dendritic cell acquires the peptide sequence invitro by exposure to a peptide comprising the peptide sequence.

In some embodiments, the peptide sequence is a synthetic peptidesequence. In some embodiments, the peptide sequence is a sequence setforth in any one of SEQ ID NO: 1-12 and SEQ ID NO: 13-17. In someinstances, the peptide sequence is a sequence set forth in SEQ ID NO:13-17 but having 1 to 4 amino acid substitutions. In certain cases, thesubstitution is at one or more of position 1, 2, or 9. In one particularembodiment, the peptide sequence is SEQ ID NO: 13. In anotherembodiment, the peptide is SEQ ID NO:13 but having 1 to 4 amino acidsubstitutions. In certain cases, the substitution is at one or more ofposition 1, 2, or 9. In another particular embodiment, the peptidesequence is SEQ ID NO: 16. In another embodiment, the peptide is SEQ IDNO:16 but having 1 to 4 amino acid substitutions. In certain cases, thesubstitution is at one or more of position 1, 2, or 9.

In some embodiments, the composition comprises between 10⁵ and 10⁸dendritic cells.

In some embodiments, the composition further comprises a peptide setforth in any one of SEQ ID NO: 1-12 and SEQ ID NO: 13-17. In oneparticular embodiment, the peptide sequence is SEQ ID NO: 13. In anotherparticular embodiment, the peptide sequence is SEQ ID NO: 16. In someembodiments, the composition comprises an agent selected from the groupconsisting of compounds to enhance the BCMA and TACT-specific responsessuch as (1) Cytokines and Chemokines; (2) checkpoint inhibitorsincluding anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, and anti-TIM3; (2)immune agonists including anti-CD28, anti-CD40L (CD154), anti-41BB(CD137), anti-OX40 and anti-GITR, (3) immune modulators includinglenalidomide, pomalidomide, a Thalidomide analogue, IMiDS compound,and/or HDAC inhibitors (e.g., ACY241) as a single agent and/or incombination with Dexamethasone; (4) adjuvant; (5) therapeutics whichincrease the BCMA and TACI-specific responses including with vaccine,cell therapies and/or antibodies; (6) therapeutics which alternate theBCMA and TACT-specific responses including peptide-based vaccine,different types of vaccine (RNA vaccine, DNA vaccine), cell therapies,specific modulators and/or specific inhibitors; and (7) therapeuticsthat have an independent approach from the BCMA and TACI-targetingtherapy to widely cover immune responses to the disease includingbiological and non-biological approaches. In some embodiments, thecomposition further comprises an immune agonist (e.g., anti-OX40antibody, anti-GITR antibody). In some embodiments, the compositionfurther comprises a checkpoint inhibitor (e.g., anti-LAG3 antibody).

In one aspect, the disclosure relates to a method of inducing an immuneresponse against BCMA- and/or TACI-expressing cell (e.g., cancer cells)in a human subject in need thereof, the method comprising administeringto the human subject a peptide as described herein (e.g., a peptidecomprising or consisting of SEQ ID NOs: 13-17), or a composition (e.g.,a pharmaceutical composition) as described herein. In anotherembodiment, the peptide is SEQ ID NO:13 but having 1 to 4 amino acidsubstitutions. In certain cases, the substitution is at one or more ofposition 1, 2, or 9 In another particular embodiment, the peptidesequence is SEQ ID NO: 16. In another embodiment, the peptide is SEQ IDNO:16 but having 1 to 4 amino acid substitutions. In certain cases, thesubstitution is at one or more of position 1, 2, or 9.

In some embodiments, the subject has a cancer that expresses BCMA and/orTACI, and the immune response is against such a cancer cell.

In some embodiments, the subject has a non-cancer cell that expressesBCMA and/or TACI, and the immune response is against such a non-cancercell.

In some embodiments, the cancer is a hematological cancer (e.g.,multiple myeloma). In some embodiments, the cancer cell is a plasma cell(e.g., cancerous plasma cells). In some embodiments, the human subjecthas refractory multiple myeloma. In some embodiments, the human subjecthas refractory multiple myeloma relapsing after allotransplantation.

In some embodiments, the cancer cell expresses BCMA, and the level ofBCMA in the cancer cell is at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 40%, or at least 50%more than a plasma cell in a healthy human subject.

In some embodiments, the cancer cell expresses TACI, and the level ofTACI in the cancer cell is at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 40%, or at least 50%more than a plasma cell in a healthy human subject.

In some embodiments, the method further comprises administering to thehuman subject a CTL specific for BCMA. In some embodiments, the methodfurther comprises administering to the human subject a CTL specific forTACI. In certain instances, the CTL is a CTL obtained by exposure to apeptide comprising or consisting of one or more of SEQ ID NO: 13 or 14.In some cases, the CTL is exposed to a peptide of SEQ ID NO: 13 or 14,but with 1 to 4 substitutions. In some cases, the substitutions are atone or more of positions 1, 2, or 9. In some cases, the peptide is 9 to30 amino acids in length. In other instances, the CTL is a CTL obtainedby exposure to a peptide comprising or consisting of any one or more ofSEQ ID NOs: 15-17. In some cases, the CTL is exposed to a peptide of SEQID NO: 15, 16, or 17, but with 1 to 4 substitutions. In some cases, thesubstitutions are at one or more of positions 1, 2, or 9. In some cases,the peptide is 9 to 30 amino acids in length. In some instances, the CTLis a memory CD8⁺ CTL. In some instances, the CTL is a memory CD8⁺CD45RO⁺ CTL. In some instances, the CTL is an effector CD8⁺ CTL. In someinstances, the CTL is an activated CD8⁺ CTL. In some instances, the CTLis a Tetramer-positive CD8⁺ CTL. In some instances, the CD8⁺ CTL is aCTL that when stimulated with a peptide described herein upregulates acostimulatory molecule expression. In some instances, the CD8⁺ CTL is aCTL that when stimulated with a peptide described herein upregulates acheckpoint molecule expression. In some instances, the CTL is a CD8⁺ CTLthat produce cytokine(s) and/or that has upregulated critical cytolyticmarker(s) (e.g. CD107, Granzyme, Perforin) expression and/or production.In some instances, the CTL is a CD8⁺ CTL that has activities againsttumor or other targets.

In some embodiments, the method further comprises administering to thehuman subject an immune agonist. In some embodiments, the immune agonistis anti-CD28, anti-CD40L (CD154), anti-41BB (CD137), anti-OX40 andanti-GITR.

In some embodiments, the method further comprises administering to thehuman subject a checkpoint inhibitor (e.g., anti-LAG3 antibody). Incertain embodiments, the method further comprises administering to thehuman subject lenalidomide. In some embodiments, the method furthercomprises administering to the human subject one or more of: an immuneagonist (e.g., anti-OX40 antibody, anti-GITR antibody), a checkpointinhibitor (e.g., anti-LAG3 antibody), or an immune modulator.

In some embodiments, the method further comprises, after administeringto the human subject the peptide or the composition, determining whetheran immune response against BCMA and/or TACI-expressing cancers occurredin the human subject.

In one aspect, the disclosure relates to a method of treating a humansubject having a cancer or a pre-malignant disease, the methodcomprising administering to the human subject a peptide as describedherein, or a composition as described herein.

In some embodiments, the cancer is a hematologic cancer. In someembodiments, the cancer is multiple myeloma, leukemia, lymphoma or anyB-cell or plasma cell malignancy.

In some embodiments, the pre-malignant disease is monoclonal gammopathyof undermined significance (MGUS) or smoldering multiple myeloma.

In some embodiments, the method further comprises detecting that one ormore cancer cells in the human subject expresses or overexpress BCMAand/or TACI.

In some embodiments, the human subject has one or more cancer cells thatoverexpress BCMA and/or TACI, wherein the level of BCMA and/or TACI inthe cancer cell is at least 20% more than a normal cell (e.g., a plasmacell in a healthy subject).

In some embodiments, the subject has one or more cancer cells thatexpress a MEW molecule.

In some embodiments, the method further comprises administering to thehuman subject a CTL specific for BCMA. In some embodiments, the methodfurther comprises administering to the human subject a CTL specific forTACI. In certain instances, the CTL is a CTL obtained by exposure to apeptide comprising or consisting of one or more of SEQ ID NO: 13 or 14.In some cases, the CTL is exposed to a peptide of SEQ ID NO: 13 or 14,but with 1 to 4 substitutions. In some cases, the substitutions are atone or more of positions 1, 2, or 9. In some cases, the peptide is 9 to30 amino acids in length. In other instances, the CTL is a CTL obtainedby exposure to a peptide comprising or consisting of any one or more ofSEQ ID NOs: 15-17. In some cases, the CTL is exposed to a peptide of SEQID NO: 15, 16, or 17, but with 1 to 4 substitutions. In some cases, thesubstitutions are at one or more of positions 1, 2, or 9. In some cases,the peptide is 9 to 30 amino acids in length. In some instances, the CTLis a memory CD8⁺ CTL. In some instances, the CTL is a memory CD8⁺CD45RO⁺ CTL. In some instances, the CTL is an effector CD8⁺ CTL. In someinstances, the CTL is activated CD8⁺ CTL. In some instances, the CTL isa Tetramer-positive CD8⁺ CTL. In some instances, the CTL is a CD8⁺ CTLthat has upregulated a costimulatory molecule expression. In someinstances, the CTL is a CD8⁺ CTL that has upregulated a checkpointmolecule expression.

In some embodiments, the method comprises administering to the humansubject an agent selected from the group consisting of compounds toenhance the BCMA and TACI-specific responses such as (1) Cytokines andChemokines; (2) checkpoint inhibitors including anti-PD1, anti-PDL1,anti-CTLA4, anti-LAG3, and anti-TIM3; (2) immune agonists includinganti-OX40 and anti-GITR, (3) immune modulators including lenalidomide,pomalidomide, HDAC inhibitors (e.g., ACY241); (4) adjuvant; (5)therapeutics which increase the BCMA and TACI-specific responsesincluding with vaccine, cell therapies and/or antibodies; and (6)therapeutics that have an independent approach from the BCMA andTACI-targeting therapy to widely cover immune responses to the disease.In some embodiments, the method further comprises administering to thehuman subject an immune agonist. In some embodiments, the immune agonistis an anti-OX40 antibody or an anti-GITR antibody.

In some embodiments, the method further comprises administering to thehuman subject a checkpoint inhibitor (e.g., anti-LAG3 antibody). Incertain embodiments, the method further comprises administering to thehuman subject lenalidomide. In some embodiments, the method furthercomprises administering to the human subject one or more of: an immuneagonist (e.g., anti-OX40 antibody, anti-GITR antibody), a checkpointinhibitor (e.g., anti-LAG3 antibody), or an immune modulator.

In some embodiments, the method further comprises administering achemotherapy or a radiotherapy to the human subject.

In one aspect, the disclosure relates to a method of generating and/orproliferating BCMA-specific cytotoxic T cells, the method comprisingcontacting one or more cytotoxic T cells with one or more antigenpresenting cells pulsed with a peptide comprising an amino acid sequenceselected from SEQ ID NO: 13 and SEQ ID NO: 14. In some cases, the CTL isexposed to a peptide of SEQ ID NO: 13 or 14, but with 1 to 4substitutions. In some cases, the substitutions are at one or more ofpositions 1, 2, or 9. In some cases, the peptide is 9 to 30 amino acidsin length.

In some embodiments, the cytotoxic T cells are memory cytotoxic T cells.In some embodiments, the cytotoxic T cells are effector cytotoxic Tcells. In some instances, the CD8+ CTL is a CTL that when stimulatedwith a peptide described herein upregulates a costimulatory moleculeexpression. In some instances, the CD8+ CTL is a CTL that whenstimulated with a peptide described herein upregulates a checkpointmolecule expression. In some instances, the CTL is a CD8+ CTL thatproduce cytokine(s) and/or that has upregulated critical cytolyticmarker(s) (e.g. CD107, Granzyme, Perform) expression and/or production.In some instances, the CTL is a CD8+ CTL that has activities againsttumor or other targets.

In some embodiments, the antigen presenting cells are dendritic cells(DCs). In one particular embodiment, the peptide comprises or consistsof SEQ ID NO: 13. In another aspect, the disclosure relates to a methodof generating TACI-specific cytotoxic T cells, the method comprisingcontacting one or more cytotoxic T cells with one or more antigenpresenting cells pulsed with a peptide comprising an amino acid sequenceselected from SEQ ID NO: 15-17. In some cases, the CTL is exposed to apeptide of SEQ ID NO: 15, 16, or 17, but with 1 to 4 substitutions. Insome cases, the substitutions are at one or more of positions 1, 2, or9. In some cases, the peptide is 9 to 30 amino acids in length.

In one particular embodiment, the peptide comprises or consists of SEQID NO: 16.

In one aspect, the disclosure also relates to a method of killing atarget cell, the method comprising contacting the target cell with oneor more BCMA-specific cytotoxic T cells, wherein the target cellexpresses or overexpresses BCMA. In some embodiments, the target cellexpresses HLA-A.

In some embodiments, the method further comprises contacting the one ormore BCMA-specific cytotoxic T cells with an agent selected from thegroup consisting of compounds to enhance the BCMA and TACI-specificresponses such as (1) Cytokines and Chemokines; (2) checkpointinhibitors including anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, andanti-TIM3; (2) immune agonists including anti-OX40 and anti-GITR, (3)immune modulators including lenalidomide, pomalidomide, a Thalidomideanalogue, IMiDS compound, and/or HDAC inhibitors (e.g., ACY241) as asingle agent and/or in combination with Dexamethasone; (4) adjuvant; (5)therapeutics which increase the BCMA and TACI-specific responsesincluding with vaccine, cell therapies and/or antibodies; and (6)therapeutics that have an independent approach from the BCMA andTACI-targeting therapy to widely cover immune responses to the disease.

In some embodiments, the method further comprises contacting the one ormore BCMA-specific cytotoxic T cells with an immune agonist.

In some embodiments, the method comprises further administering apeptide comprising or consisting of an amino acid sequence set forth inSEQ ID NOs: 13 or 14. In some cases, the peptide may have 1 to 15 (i.e.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acids added atthe N- and/or C-terminus of the amino acid sequence of SEQ ID NO.: 13 or14.

In some embodiments, the immune agonist is an OX40 agonist or an GITRagonist. In some embodiments, the immune agonist is an anti-OX40antibody or an anti-GITR antibody.

In some embodiments, the method further comprises administering to thehuman subject a checkpoint inhibitor (e.g., anti-LAG3 antibody). Incertain embodiments, the method further comprises administering to thehuman subject lenalidomide. In some embodiments, the method furthercomprises administering to the human subject one or more of: an immuneagonist (e.g., anti-OX40 antibody, anti-GITR antibody), a checkpointinhibitor (e.g., anti-LAG3 antibody), or lenalidomide.

In another aspect, the disclosure relates to a method of killing atarget cell, the method comprising contacting the target cell with oneor more TACI-specific cytotoxic T cells, wherein the target cellexpresses or overexpresses TACI. In some embodiments, the target cellexpresses HLA-A.

In some embodiments, the method comprises further administering apeptide comprising or consisting of an amino acid sequence set forth inany one of SEQ ID NOs: 15-17. In some cases, the peptide may have 1 to15 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) amino acidsadded at the N- and/or C-terminus of the amino acid sequence of SEQ IDNO.: 15-17.

In some embodiments, the method further comprises contacting the one ormore TACI-specific cytotoxic T cells with an agent selected from thegroup consisting of compounds to enhance the BCMA and TACI-specificresponses such as (1) Cytokines and Chemokines; (2) checkpointinhibitors including anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, andanti-TIM3; (2) immune agonists including anti-OX40 and anti-GITR, (3)immune modulators including lenalidomide, pomalidomide, HDAC inhibitors(e.g., ACY241); (4) adjuvant; (5) therapeutics which increase the BCMAand TACI-specific responses including with vaccine, cell therapiesand/or antibodies; and (6) therapeutics that have an independentapproach from the BCMA and TACI-targeting therapy to widely cover immuneresponses to the disease.

In some embodiments, the method further comprises contacting the one ormore TACI-specific cytotoxic T cells with an immune agonist.

In some embodiments, the immune agonist is an OX40 agonist or an GITRagonist.

In some embodiments, the immune agonist is an anti-OX40 antibody or ananti-GITR antibody. In some embodiments, the method further comprisesadministering to the human subject a checkpoint inhibitor (e.g.,anti-LAG3 antibody). In certain embodiments, the method furthercomprises administering to the human subject lenalidomide. In someembodiments, the method further comprises administering to the humansubject one or more of: an immune agonist (e.g., anti-OX40 antibody,anti-GITR antibody), a checkpoint inhibitor (e.g., anti-LAG3 antibody),or lenalidomide.

In one aspect, the disclosure relates to a method of treating a humansubject having a BCMA or TACI-expressing disease or cancer, the methodcomprising administering a plurality of BCMA-specific cytotoxic T cellsor TACI-specific cytotoxic T cells to the human subject.

In some embodiments, the method further comprises administering to thehuman subject a peptide comprising or consisting of a sequence set forthin any one of SEQ ID NOs: 13-17. In some instances, the peptide has asequence set forth in one of SEQ ID NO: 13-17 except that it has 1 to 4substitutions. In some cases, the substitutions may be at one or more ofpositions 1, 2, or 9. In some instances, the peptide is 9 to 30 aminoacids in length.

In some embodiments, the method further comprises administering to thehuman subject an agent selected from the group consisting of compoundsto enhance the BCMA and TACI-specific responses such as (1) Cytokinesand Chemokines; (2) checkpoint inhibitors including anti-PD1, anti-PDL1,anti-CTLA4, anti-LAG3, and anti-TIM3; (2) immune agonists includinganti-OX40 and anti-GITR, (3) immune modulators including lenalidomide,pomalidomide, HDAC inhibitors (e.g., ACY241); (4) adjuvant; (5)therapeutics which increase the BCMA and TACI-specific responsesincluding with vaccine, cell therapies and/or antibodies; and (6)therapeutics that have an independent approach from the BCMA andTACI-targeting therapy to widely cover immune responses to the disease.

In some embodiments, the method further comprises administering to thesubject an immune agonist. In some embodiments, the immune agonist is anOX40 agonist or an GITR agonist. In some embodiments, the immune agonistis an anti-OX40 antibody or an anti-GITR antibody.

In some embodiments, the method further comprises administering to thehuman subject a checkpoint inhibitor (e.g., anti-LAG3 antibody). Incertain embodiments, the method further comprises administering to thehuman subject lenalidomide. In some embodiments, the method furthercomprises administering to the human subject one or more of: an immuneagonist (e.g., anti-OX40 antibody, anti-GITR antibody), a checkpointinhibitor (e.g., anti-LAG3 antibody), or lenalidomide.

In some embodiments, the cytotoxic T cells are derived from the cells ofthe human subject. In some embodiments, the cytotoxic T cells arederived from induced pluripotent stem cells.

In some embodiments, the cancer expresses BCMA and/or TACI. In someembodiments, the human subject has multiple myeloma.

In one aspect, the disclosure relates to a process comprising,

(a) obtaining bone marrow derived mononuclear cells from a subject;

(b) culturing the mononuclear cells in vitro under a condition in whichmononuclear cells become adherent to a culture vessel;

(c) selecting adherent mononuclear cells;

(d) culturing the adherent mononuclear cells in the presence of one ormore cytokines under a condition in which the cells differentiate intoantigen present cells; and

(e) contacting the antigen presenting cells with a peptide as describedherein (e.g., SEQ ID NOs: 13-17), thereby generating antigen presentingcells that present the peptide on a major histocompatibility complex(MHC) molecule.

In some embodiments, the major histocompatibility complex molecule is aMHC class I molecule.

In some embodiments, the one or more cytokines comprise granulocytemacrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4).

In some embodiments, the one or more cytokines comprise tumor necrosisfactor-α (TNF-α).

In some embodiments, the bone marrow derived cells are obtained from asubject diagnosed with multiple myeloma.

In one aspect, the discourse relates to a method of identifying a T cellantigen receptor sequence for BCMA, the method comprising

(a) generating and/or proliferating BCMA-specific cytotoxic T cells bythe method as described herein;

(b) determining the T cell antigen receptor sequence for BCMA in theBCMA-specific cytotoxic T cells.

In another aspect, the disclosure relates to a method for treating ahuman subject having a BCMA-expressing disease or cancer, comprising:administering to the human subject a composition comprising a chimericantigen receptor T cell (CAR-T cell), wherein the CAR-T cell expresses achimeric antigen receptor, wherein the chimeric antigen receptor bindsto BCMA.

In some embodiments, the cancer expresses BCMA. In some embodiments, thehuman subject has multiple myeloma.

In some embodiments, the method further comprises administering to thehuman subject a peptide comprising or consisting of a sequence set forthin any one of SEQ ID NO.: 13 or 14. In some cases, the peptide has thesequence set forth in SEQ ID NO:13 or 14, except that it has 1 to 4amino acid substitutions. In some instances, the substitutions are atone or more of positions 1, 2, or 9. In some instances, the peptide is 9to 30 amino acids in length. In some embodiments, the method furthercomprises administering to the human subject an agent selected from thegroup consisting of compounds to enhance the BCMA and TACI-specificresponses such as (1) Cytokines and Chemokines; (2) checkpointinhibitors including anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, andanti-TIM3; (2) immune agonists including anti-OX40 and anti-GITR, (3)immune modulators including lenalidomide, pomalidomide, HDAC inhibitors(e.g., ACY241); (4) adjuvant; (5) therapeutics which increase the BCMAand TACI-specific responses including with vaccine, cell therapiesand/or antibodies; and (6) therapeutics that have an independentapproach from the BCMA and TACI-targeting therapy to widely cover immuneresponses to the disease.

In some embodiments, the method further comprises administering to thesubject an immune agonist. In some embodiments, the immune agonist is anOX40 agonist or an GITR agonist. In some embodiments, the immune agonistis an anti-OX40 antibody or an anti-GITR antibody.

In some embodiments, the method further comprises administering to thehuman subject a checkpoint inhibitor (e.g., anti-LAG3 antibody). Incertain embodiments, the method further comprises administering to thehuman subject lenalidomide. In some embodiments, the method furthercomprises administering to the human subject one or more of: an immuneagonist (e.g., anti-OX40 antibody, anti-GITR antibody), a checkpointinhibitor (e.g., anti-LAG3 antibody), or lenalidomide.

In one aspect, the discourse relates to a method of identifying a T cellantigen receptor sequence for TACI, the method comprising

(a) generating and/or proliferating TACI-specific cytotoxic T cells bythe method as described herein;

(b) determining the T cell antigen receptor sequence for TACI in theTACT-specific cytotoxic T cells.

In another aspect, the disclosure relates to a method for treating ahuman subject having a TACI-expressing disease or cancer, comprising:administering to the human subject a composition comprising a chimericantigen receptor T cell (CAR-T cell), wherein the CAR-T cell expresses achimeric antigen receptor, wherein the chimeric antigen receptor bindsto TACI.

In some embodiments, the cancer expresses TACI. In some embodiments, thehuman subject has multiple myeloma.

In some embodiments, the method further comprises administering to thehuman subject a peptide comprising or consisting of a sequence set forthin any one of SEQ ID NOs: 15-17.

In some embodiments, the method further comprises administering to thehuman subject an agent selected from the group consisting of compoundsto enhance the BCMA and TACI-specific responses such as (1) Cytokinesand Chemokines; (2) checkpoint inhibitors including anti-PD1, anti-PDL1,anti-CTLA4, anti-LAG3, and anti-TIM3; (2) immune agonists includinganti-OX40 and anti-GITR, (3) immune modulators including lenalidomide,pomalidomide, HDAC inhibitors (e.g., ACY241); (4) adjuvant; (5)therapeutics which increase the BCMA and TACI-specific responsesincluding with vaccine, cell therapies and/or antibodies; and (6)therapeutics that have an independent approach from the BCMA andTACI-targeting therapy to widely cover immune responses to the disease.

In some embodiments, the method further comprises administering to thesubject an immune agonist. In some embodiments, the immune agonist is anOX40 agonist or an GITR agonist. In some embodiments, the immune agonistis an anti-OX40 antibody or an anti-GITR antibody.

In some embodiments, the method further comprises administering to thehuman subject a checkpoint inhibitor (e.g., anti-LAG3 antibody). Incertain embodiments, the method further comprises administering to thehuman subject lenalidomide. In some embodiments, the method furthercomprises administering to the human subject one or more of: an immuneagonist (e.g., anti-OX40 antibody, anti-GITR antibody), a checkpointinhibitor (e.g., anti-LAG3 antibody), or lenalidomide.

In one aspect, the disclosure provides a composition comprising ananoparticle, and a peptide comprising a sequence that is at least 60%identical to any one of SEQ ID NOs: 1-17. In some embodiments, thepeptide comprises or consists of the sequence of SEQ ID NO: 13. Incertain instances, the peptide has the sequence of SEQ ID NO:13 but with1 to 4 amino acid substitutions. In some cases, the substitutions are atone or more of positions 1, 2, or 9. The peptide can be 9 to 30 aminoacids in length. In other embodiments, the sequence comprises orconsists of the sequence of SEQ ID NO: 16. In certain instances, thepeptide has the sequence of SEQ ID NO:16 but with 1 to 4 amino acidsubstitutions. In some cases, the substitutions are at one or more ofpositions 1, 2, or 9. The peptide can be 9 to 30 amino acids in length.

In some embodiments, the peptide is encapsulated in the nanoparticle. Insome embodiments, the nanoparticle is a liposome.

In some embodiments, the nanoparticle comprises a biodegradable polymer.In some embodiments, the nanoparticle comprisespoly(D,L-lactide-co-glycolide) (PLGA). In some embodiments, thenanoparticle comprises poly(lactic-co-glycolic acid)-poly(ethyleneglycol) (PLGA-PEG) copolymer.

In some embodiments, the amino acid sequence is SEQ ID NO: 13. In someembodiments, the amino acid sequence is SEQ ID NO: 14. In someembodiments, the amino acid sequence is SEQ ID NO: 15. In someembodiments, the amino acid sequence is SEQ ID NO: 16. In someembodiments, the amino acid sequence is SEQ ID NO: 17. In certaininstances, the peptide has the sequence of any one of SEQ ID NO:13 to 17but with 1 to 4 amino acid substitutions. In some cases, thesubstitutions are at one or more of positions 1, 2, or 9. The peptidecan be 9 to 30 amino acids in length.

In some embodiments, the nanoparticle comprises an adjuvant. In someembodiments, the nanoparticle comprises a Toll-like receptor agonist(e.g., R848 or unmethylated CpG oligodeoxynucleotide).

In one aspect, the disclosure provides methods for treating a humansubject having a cancer. The methods involve administering to the humansubject the composition as described herein (e.g., nanoparticles). Insome embodiments, the human subject has multiple myeloma.

In some embodiments, the cancer expresses BCMA. In other embodiments,the cancer expresses TACI.

In some embodiments, the method further comprises administering to thehuman subject a peptide comprising or consisting of a sequence set forthin any one of SEQ ID NOs: 15-17. In some embodiments, the peptide has asequence set forth in one of SEQ ID NOs:15-17 except having 1 to 4 aminoacid substitutions. The substitutions may be at one or more of positions1, 2, or 9. The peptide can be 9 to 30 amino acids in length. In someembodiments, the method further comprises administering to the humansubject a CTL specific for BCMA. In some embodiments, the method furthercomprises administering to the human subject a CTL specific for TACI. Incertain instances, the CTL is a CTL obtained by exposure to a peptidecomprising or consisting of one or more of SEQ ID NO: 13 or 14. In otherinstances, the CTL is a CTL obtained by exposure to a peptide comprisingor consisting of any one or more of SEQ ID NOs: 15-17. In someinstances, the CTL is a memory CD8+ CTL. In some instances, the CTL is amemory CD8+ CD45RO+ CTL. In some instances, the CTL is an effector CD8+CTL. In some instances, the CTL is activated CD8+ CTL. In someinstances, the CTL is a Tetramer-positive CD8+ CTL. In some instances,the CTL is a CD8+ CTL that has upregulated a costimulatory moleculeexpression. In some instances, the CTL is a CD8+ CTL that hasupregulated a checkpoint molecule expression. In some embodiments, themethod further comprises administering to the subject an immune agonist.In some embodiments, the immune agonist is an OX40 agonist or an GITRagonist. In some embodiments, the immune agonist is an anti-OX40antibody or an anti-GITR antibody.

In some embodiments, the method further comprises administering to thehuman subject a checkpoint inhibitor (e.g., anti-LAG3 antibody). Incertain embodiments, the method further comprises administering to thehuman subject lenalidomide. In some embodiments, the method furthercomprises administering to the human subject one or more of: an immuneagonist (e.g., anti-OX40 antibody, anti-GITR antibody), a checkpointinhibitor (e.g., anti-LAG3 antibody), or lenalidomide.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1I show BCMA expression on multiple myeloma cell lines.

FIG. 2 shows binding affinity of native BCMA peptides to HLA-A2.

FIG. 3 shows binding affinity of native TACI peptides to HLA-A2

FIG. 4 shows binding affinity of BCMA peptides to HLA-A2: native peptidevs. heteroclitic peptide.

FIG. 5 shows binding affinity of TACI peptides to HLA-A2: native peptidevs. heteroclitic peptide.

FIG. 6 shows HLA-A2 stability of BCMA #4 and #5 peptides: native peptidevs. heteroclitic peptide (50 ug/ml).

FIG. 7 shows HLA-A2 stability of TACI #1, #3 and #4 peptides: nativepeptide vs. heteroclitic peptide (50 ug/ml).

FIGS. 8A-8C show increased CD8⁺ cytotoxic T cell (CTL) with heterocliticBCMA #4 peptide stimulation.

FIGS. 9A-9C show decreased naïve CTL with heteroclitic BCMA #4 peptidestimulation.

FIGS. 10A-10C show increased memory CTL with heteroclitic BCMA #4peptide stimulation.

FIGS. 11A-11C show kinetics of CM vs. effector cells with heterocliticBCMA #4 peptide stimulation.

FIG. 12 shows induction of memory CD8⁺ CTL by heteroclitic BCMA #4peptide.

FIG. 13 shows induction of memory CD8⁺ CTL by heteroclitic TACI #3peptide.

FIG. 14 shows anti-tumor activities of heteroclitic BCMA #4 peptide-CTL(N=5).

FIG. 15 shows anti-tumor activities of heteroclitic TACI #3 peptide-CTL(n=4).

FIG. 16 shows HLA-A2 specific proliferation of heteroclitic BCMA #4peptide-CTL.

FIG. 17 shows enhanced α-tumor activities by central memory cells ofBCMA-specific CTL treated w. α-OX40 or α-GITR.

FIGS. 18A-18C show upregulation of critical T cells markers on BCMApeptide-specific CTL stimulated with heteroclitic BCMA peptides.

FIGS. 19A-19F show HLA-A2 restricted and antigen-specific immuneresponses by heteroclitic BCMA₇₂₋₈₀-specific CTL to HLA-A2⁺ MM celllines.

FIGS. 20A-20H show anti-tumor activities of heterocliticBCMA₅₄₋₆₂-specific CTL or heteroclitic BCMA₇₂₋₈₀ specific CTL againstpatients' MM cells.

FIGS. 21A-21C. BCMA₇₂₋₈₀ specific Tetramer⁺ CTL displaying distinctphenotypes and high level of anti-tumor activities against MM cells.

FIGS. 22A-22E. Differentiation of memory CD8⁺ T cell of BCMA-specificCTL upon the stimulation with heteroclitic BCMA₇₂₋₈₀ peptide.

FIGS. 23A-23C. Characterization of high anti-tumor activities by BCMAspecific memory CTL (FIG. 23A) and the highest levels by central memoryCTL (FIG. 23B, FIG. 23C).

FIG. 24A. The results of BCMA peptide-specific CTL co-cultured (7 days)with 25 U266 cells.

FIGS. 24B-24C. Enhanced anti-myeloma activities of memory CD8⁺ T cellsof heteroclitic BCMA72-80 CTL [generated from one HLA-A2+ individual] intreatment with anti-LAG3 or anti-OX40.

FIG. 24D. Enhanced anti-tumor activities of heteroclitic BCMA₇₂₋₈₀ CTL[generated from HLA-A2⁺ Donor 1, Donor 2 or Donor 3] in treatment withanti-OX40 against myeloma cells in an HLA-A2-restricted manner.

FIGS. 25A-25B. The percentage of CD3+CD8+ T cells after peptidestimulation with heteroclitic BCMA₇₂₋₈₀.

FIGS. 26A-26B. The percentage of CD3+CD4+ T cells after peptidestimulation with heteroclitic BCMA₇₂₋₈₀.

FIGS. 27A-27C show high BCMA expression on H929, MMIS, U266 and OPM1cell lines, but not on breast cancer cell line (MDA-MB231).

FIGS. 28A-28B show percentage of CD3+CD8+ T cells that express PD-1 andLAG-3 after peptide stimulation with heteroclitic BCMA₇₂₋₈₀.

FIGS. 29A-29C show differentiation of naïve cells into memory CTL uponstimulation with heteroclitic TACI₁₅₄₋₁₆₂ Peptide.

FIGS. 30A-30F show anti-tumor activities of heteroclitic TACI₁₅₄₋₁₆₂specific CTL against HLA-A2+ multiple myeloma cells (McCAR).

FIGS. 31A-31E show anti-tumor activities of heteroclitic TACI₁₅₄₋₁₆₂specific CTL against HLA-A2+ multiple myeloma cells.

FIG. 31F shows induction of peptide-specific Tetramer+ CTL andanti-tumor activity and proliferation by heteroclitic TACI₁₅₄₋₁₆₂peptide to HLA-A2+ multiple myeloma cells.

FIG. 32A shows morphology of BCMA Peptide loaded PLGA nanoparticles.

FIG. 32B shows morphology of BCMA Peptide loaded Liposome nanoparticles.

FIG. 32C shows BCMA peptide quantification.

FIG. 33A shows a higher loading efficiency of BCMA peptide on dendriticcells, upon encapsulation in nanoparticle (PLGA, Liposome), in atime-dependent manner.

FIG. 33B shows a higher loading efficiency of BCMA peptide-FITC ondendritic cells, upon encapsulation in nanoparticle (PLGA, Liposome), ina time-dependent manner.

FIG. 33C shows higher PLGA/peptide uptake by dendritic cells.

FIG. 33D shows a higher uptake of BCMA peptide-FITC by dendritic cells,upon encapsulation in PLGA, in a time-dependent manner.

FIG. 33E shows a higher uptake of BCMA peptide-FITC by T2 cells, uponencapsulation in PLGA, in a time-dependent manner.

FIG. 34A shows the highest anti-MM activities by BCMA-CTL generated withPLGA/BCMA peptide against MM cell lines in an HLA-A2-restricted manner.

FIG. 34B shows the highest IFN-γ production by BCMA-CTL generated withPLGA/BCMA peptide against MM cell lines in an HLA-A2-restricted manner.

FIG. 34C shows the highest IL-2 production by BCMA-CTL generated withPLGA/BCMA peptide against MM cell lines in an HLA-A2-restricted manner.

FIG. 34D shows the highest TNF-α production by BCMA-CTL generated withPLGA/BCMA peptide against MM cell lines in an HLA-A2-restricted manner.

FIG. 34E shows the highest anti-MM activities and Th1 type cytokines(IFN-γ, IL-2, TNF-α) production by BCMA-CTL generated from differentHLA-A2⁺ individuals (N=3) with PLGA/BCMA peptide against MM cell linesin an HLA-A2-restricted manner.

FIG. 35A shows the baseline activities of BCMA-specific CTL generatedwith BCMA peptide itself, PLGA/BCMA peptide or Liposome/BCMA peptide, inthe absence of tumor cells.

FIG. 35B shows the highest anti-MM activities by BCMA-CTL generated withPLGA/BCMA peptide in response to primary HLA-A2⁺ CD138⁺ tumor cells fromHLA-A2⁺ myeloma patient #1.

FIG. 35C shows the highest anti-MM activities by BCMA-CTL generated withPLGA/BCMA peptide in response to primary HLA-A2⁺ CD138⁺ tumor cells fromHLA-A2⁺ myeloma patient #2.

FIG. 35D shows the highest anti-MM activities and Th1 type cytokines(IFN-γ, IL-2, TNF-α) production by BCMA-CTL generated with PLGA/BCMApeptide against myeloma patients' tumor cells in an HLA-A2-restrictedmanner.

FIG. 36A shows a higher frequency of Tetramer⁺ CD8⁺ T cells andcostimulatory molecule expressing (CD28⁺) CD8⁺ T cells by BCMA-CTLgenerated with PLGA/BCMA peptide than with BCMA peptide itself.

FIG. 36B shows a higher increase of peptide-specific CD8⁺ T cellsproliferation by BCMA-CTL generated with PLGA/BCMA peptide than withBCMA peptide itself.

FIG. 36C shows a higher increase of peptide-specific IFN-γ production byBCMA-CTL generated with PLGA/BCMA peptide than with BCMA peptide itself.

FIG. 37A shows the highest induction of myeloma-specific CD8⁺ T cellsproliferation by BCMA-CTL generated with PLGA/BCMA peptide than withBCMA peptide itself.

FIG. 37B shows a higher increase in CD45RO+ memory/CD3⁺ CD8⁺ T cellssubset in BCMA-CTL [representative results], upon repeated stimulationwith PLGA/BCMA peptide than with BCMA peptide itself.

FIG. 37C shows a higher increase in CD45RO+ memory/CD3⁺ CD8⁺ T cellssubset in BCMA-CTL [N=3 results], upon repeated stimulation withPLGA/BCMA peptide than with BCMA peptide itself.

FIG. 37D shows a higher induction and maintenance of central memory/CD3⁺CD8⁺ T cells subset in BCMA-CTL, upon repeated stimulation withPLGA/BCMA peptide than with BCMA peptide itself.

FIG. 38A shows a higher induction of central memory and effector memoryCTL and their anti-MM activities against myeloma cells, in anHLA-A2-restricted manner, by BCMA-CTL generated with PLGA/BCMA peptidethan with BCMA peptide itself.

FIG. 38B shows a higher induction of central memory and effector memoryCTL and their IFN-γ production against myeloma cells, in anHLA-A2-restricted manner, by BCMA-CTL generated with PLGA/BCMA peptidethan with BCMA peptide itself.

FIG. 38C shows a higher induction of central memory and effector memoryCTL and their anti-MM activities and IFN-γ, IL-2, TNF-α productionagainst myeloma cells, in an HLA-A2-restricted manner, by BCMA-CTLgenerated with PLGA/BCMA peptide than with BCMA peptide itself.

DETAILED DESCRIPTION

B-cell maturation antigen (BCMA) and Transmembrane activator and CAMLinteractor (TACI) are critical antigens specific to many cancers,including, e.g., multiple myeloma and other hematological malignancies.This disclosure is based at least, in part, on the identification ofHLA-A2-specific immunogenic peptides derived from BCMA and TACIantigens, which can be used to generate, e.g., multiple myeloma(MM)-specific T cells immune response. Thus, the disclosure relates toBCMA-derived peptides and TACI-derived peptides (and pharmaceuticalcompositions thereof), which can be used to, e.g., induce an immuneresponse (e.g., stimulate a cytotoxic T cell (CTL) response) againsttumor cells, or stimulate the production of cytokines or antibodies, ina subject. The peptides can be used in a variety of applications such asmethods for inducing an immune response, methods for producing anantibody, methods for producing cytokines involved in anti-tumor immuneresponses, and methods for treating a cancer (e.g., such as multiplemyeloma). The peptides can also be included in MHC molecule multimercompositions and used in, e.g., methods for detecting a T cell (e.g.,the antigen-specific T cell) in a population of cells.

The present disclosure also provides information for various types oftherapeutic applications including peptide-based vaccination, peptidecomposing various approaches of vaccine (including nanoparticle-basedand virus-based), adoptive transfer of ex vivo generated BCMA-specific Tcells or TACI-specific T cells and the antigen-specific T cells withengineered technology (including CAR-TCR therapy-based and inducedpluripotent stem cell-based) or infusion of peptide-pulsed dendriticcells, as a single or combination therapy described herein, in cancerpatients including multiple myeloma, their pre-malignant diseases orother cancers, or any diseases which uniquely-express and/or overexpressthe BCMA and TACI antigens.

BCMA-Derived Peptides and TACI-Derived Peptides

B-cell maturation antigen (BCMA) (NM_001192.2 ΘNP_001183.2), also knownas tumor necrosis factor receptor superfamily member 17 (TNFRSF17), is aprotein that in humans is encoded by the TNFRSF17 gene. BCMA is a cellsurface receptor of the TNF receptor superfamily which recognizes B-cellactivating factor (BAFF). BCMA is expressed in mature B lymphocytes.This receptor has been shown to specifically bind to the tumor necrosisfactor (ligand) superfamily, member 13b (TNFSF13B/TALL-1/BAFF), and tolead to NF-kappaB and MAPK8/JNK activation. This receptor also binds tovarious TRAF family members, and thus may transduce signals for cellsurvival and proliferation. BCMA is often overexpressed in variouscancer cells, e.g., in a subject with leukemia, lymphomas, and multiplemyeloma.

Transmembrane activator and CAML interactor (TACI)(NM_012452.2→NP_036584.1), also known as tumor necrosis factor receptorsuperfamily member 13B (TNFRSF13B) is a protein that in humans isencoded by the TNFRSF13B gene. TACI is a transmembrane protein of theTNF receptor superfamily found predominantly on the surface of B cells.TACI recognizes three ligands: APRIL, BAFF and CAML. TACI is also oftenoverexpressed in various cancer cells as well, e.g., in a subject withleukemia, lymphomas, and multiple myeloma.

The amino acid sequences of human BCMA and human TACI are shown below.

Human BCMA (NP_001183.2; SEQ ID NO: 18)

1 mlqmagqcsq neyfdsllha cipcqlrcss ntppltcqry cnasvtnsvk gtnailwtcl61 glsliislav fvlmfllrki nseplkdefk ntgsgllgma nidleksrtg deiilprgle121 ytveectced cikskpkvds dhcfplpame egatilvttk tndyckslpa alsateieks181 isar

Human TACI (NP_036584.1; SEQ ID NO: 19)

1 msglgrsrrg grsrvdqeer fpqglwtgva mrscpeeqyw dpllgtcmsc kticnhqsqr61 tcaafcrsls crkeqgkfyd hllrdcisca sicgqhpkqc ayfcenklrs pvnlppelrr121 qrsgevenns dnsgryqgle hrgseaspal pglklsadqv alvystlglc lcavlccflv181 avacflkkrg dpcscqprsr prqspakssq dhameagspv stspepvetc sfcfpecrap241 tqesavtpgt pdptcagrwg chtrttvlqp cphipdsglg ivcvpaqegg pga

The disclosure provides peptides (e.g., naïve peptides) that are derivedfrom BCMA or TACI antigen and heteroclitic peptide that are derived fromthe peptides (naïve peptides). These peptides can be any portion orfragment of the BCMA or TACI peptide. In some embodiments, thesepeptides have a length of greater than 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30amino acid residues. In some embodiments, these peptides have a lengthof less than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues. In someembodiments, these peptides have a length of 8 to 12 amino acidresidues, 8 to 15 amino acid residues, 9 to 13 amino acid residues, 9 to12 amino acid residues, or 11 to 30 amino acid residues. In someembodiments, the length of the peptide is 9, 10, 11, or 12 amino acidresidues (e.g., 9 amino acid residues).

In some embodiments, peptides that are derived from BCMA or TACI includethe following:

#1. BCMA₆₄₋₇₂ (SEQ ID NO: 1) (LIISLAVFV) #2. BCMA₆₉₋₇₇ (SEQ ID NO: 2)(AVFVLMFLL) #3. BCMA₉₋₁₇ (SEQ ID NO: 3) (SQNEYFDSL) #4. BCMA₇₂₋₈₀(SEQ ID NO: 4) (VLMFLLRKI) #5. BCMA₅₄₋₆₂ (SEQ ID NO: 5) (AILWTCLGL)#6. BCMA₁₁₄₋₁₂₂ (SEQ ID NO: 6) (ILPRGLEYT) #1. TACI₁₇₈₋₁₈₆(SEQ ID NO: 7) (FLVAVACFL) #2. TACI₁₇₄₋₁₈₂ (SEQ ID NO: 8) (VLCCFLVAV)#3. TACI₁₅₄₋₁₆₂ (SEQ ID NO: 9) (KLSADQVAL) #4. TACI₁₆₆₋₁₇₄(SEQ ID NO: 10) (TLGLCLCAV) #5. TACI₁₆₁₋₁₆₉ (SEQ ID NO: 11) (ALVYSTLGL)#6. TACI₁₅₅₋₁₆₃ (SEQ ID NO: 12) (LSADQVALV)

These peptides can bind to Major Histocompatibility Complex (MHC)molecules. MHC is a large gene family with an important role in theimmune system, autoimmunity, and reproduction. MHC molecules assumeroles in the presentation of peptides, including self and non-self(antigenic) on their surface to T-cells. MHC class I molecules bindshort peptides, whose N- and C-terminal ends are anchored into thepockets located at the ends of the peptide binding groove. While many ofthese peptides are of length 9, longer peptides can be accommodated bythe bulging of their central portion, resulting in binding peptides oflength, e.g., 8 to 15. Peptides binding to class II proteins are notconstrained in size and can vary, e.g., from 11 to 30 amino acids long.The peptide binding groove in the MHC class II molecules is open at bothends, which enables binding of peptides with relatively longer length.Though the “core” nine residues long segment contributes the most to therecognition of the peptide, the flanking regions are also important forthe specificity of the peptide to the class II allele.

Thus, the disclosure also provides a peptide that has a sequence thatcomprises, consists of, or consists essentially of any sequences thatare described in the present disclosure. In some embodiments, thepeptides can bind to MHC class I molecules and/or MHC class IImolecules. In some embodiments, the MHC class I molecule is HLA-A (e.g.,HLA-A2, HLA-A24, HLA-A1, HLA-A3, HLA-A30, HLA-A26, HLA-A68, or HLA-A11),HLA-B or HLA-C.

In order to improve the stability of the peptide binding to MHCmolecules (e.g., MHC class I molecules, HLA-A2) molecules, increaseimmunogenicity, and/or increase immune response, various modificationscan be made to the peptides. For example, in order to increaseimmunogenicity, amino acid residues can be modified, by enhancingaffinity to the T cell receptor (TCR) by altering TCR interaction sites,e.g., in the positions 3, 4, 5, 6, 7, and/or 8 of any peptides describedherein (e.g., SEQ ID NOS: 1-17).

Dibasic amino acid residues (e.g., Arg-Arg, Arg-Lys, Lys-Arg, orLys-Lys) can also be added to the N- and C-termini of peptides. In someembodiments, amino acids are substituted either to enhance MajorHistocompatibility Complex (WIC) binding by modifying anchor residues(“fixed anchor epitopes”) or to enhance binding to the T cell receptor(TCR) by modifying TCR interaction sites (e.g., positions 1, 2, and/or 9of SEQ ID NOS: 1-17). In some embodiments, the epitopes described hereincan be modified at any position (e.g., at one, two, three, four, five,or six positions). The peptides can also include internal mutations thatrender them “superantigens” or “superagonists” for T cell stimulation.Superantigen peptides can be generated by screening T cells with apositional scanning synthetic peptide combinatorial library (PS-CSL) asdescribed in Pinilla et al, Biotechniques, 13(6): 901-5, 1992; Borras etal, J. Immunol. Methods, 267(1): 79-97, 2002; U.S. Publication No.2004/0072246; and Lustgarten et al., J. Immun. 176: 1796-1805, 2006. Insome embodiments, a superagonist peptide is a peptide as describedherein, with one, two, three, or four amino acid substitutions whichrender the peptide a more potent immunogen. In some embodiments, thefirst amino acid residues of the peptides that are derived from BCMA orTACI can be changed to Tyrosine.

Some heteroclitic peptides that are derived from BCMA or TACI include:

Heteroclitic #4. BCMA₇₂₋₈₀ (SEQ ID NO: 13) (YLMFLLRKI)Heteroclitic #5. BCMA₅₄₋₆₂ (SEQ ID NO: 14) (YILWTCLGL)Heteroclitic #1. TACI₁₇₈₋₁₈₆ (SEQ ID NO: 15) (YLVAVACFL)Heteroclitic #3. TACI₁₅₄₋₁₆₂ (SEQ ID NO: 16) (YLSADQVAL)Heteroclitic #4. TACI₁₆₆₋₁₇₄ (SEQ ID NO: 17) (YLGLCLCAV)

As used herein, the term “heteroclitic” (e.g., a heteroclitic peptide)refers to a form of a peptide in which one or more amino acid residueshave been modified from a wild-type or original sequence in order toproduce a peptide that is more immunogenic than the correspondingpeptide with wildtype sequence or original sequence.

The disclosure further provides variants of the peptides as describedherein (e.g., SEQ ID NO: 1-17). Variants of the peptides describedherein can include forms of the peptides having not more than five, notmore than four, not more than three, not more than two, not more thanone amino acid substitutions (e.g., 5, 4, 3, 2, or 1 amino acidsubstitutions). In some embodiments, variants of the peptides describedherein can include forms of the peptides having at least one, at leasttwo, at least three, or at least four substitutions.

In some embodiments, amino acids at positions 1, 2, and/or 9 of SEQ IDNOS: 1-17 contribute to HLA binding affinity, and thus can besubstituted without affecting the peptide-specific T cells responses.Thus, the immunogenicity of peptides can be still maintained withsubstitutions at position of 1, 2 and/or 9 of SEQ ID NOS: 1-17. In someembodiments, the amino acid at position 1 of SEQ ID NOS: 1-17 (e.g., SEQID NO: 13 or SEQ ID NO: 16) is substituted. In some embodiments, theamino acid at position 2 of SEQ ID NOS: 1-17 (e.g., SEQ ID NO: 13 or SEQID NO: 16) is substituted. In some embodiments, the amino acid atposition 9 of SEQ ID NOS: 1-17 (e.g., SEQ ID NO: 13 or SEQ ID NO: 16) issubstituted. In some embodiments, the amino acids at positions 1 and 2,positions 1 and 9, positions 2 and 9, or positions 1, 2, and 9 of SEQ IDNOS: 1-17 are substituted. In some embodiments, the disclosure providesa peptide comprising a sequence as described herein (e.g., one of SEQ IDNOS: 1-17 with or without substitutions as described herein) and has upto 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 amino acids.Thus, in some embodiments, the length of the peptide can be between 9and 20 amino acids, between 9 and 30 amino acids, or between 9 and 40amino acids.

The substitutions can be any type of amino acid substitution, e.g.,conservative or non-conservative. Conservative substitutions includesubstitutions within the following groups: (1) valine, alanine andglycine; leucine, valine, and isoleucine; (2) aspartic acid and glutamicacid; (3) asparagine and glutamine; (4) serine, cysteine, and threonine;lysine and arginine; and (5) phenylalanine and tyrosine. The non-polarhydrophobic amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan and methionine. The polar neutralamino acids include glycine, serine, threonine, cysteine, tyrosine,asparagine and glutamine. The positively charged (basic) amino acidsinclude arginine, lysine, and histidine. The negatively charged (acidic)amino acids include aspartic acid and glutamic acid. Any substitution ofone member of the above-mentioned polar, basic or acidic groups byanother member of the same group can be deemed a conservativesubstitution. By contrast, a non-conservative substitution is asubstitution of one amino acid for another with dissimilarcharacteristics, e.g., substituting an amino acid with another aminoacid within another group.

In some embodiments, one or more (e.g., one, two, three, four, or allfive) of positions three, four, five, six, seven, and eight of any ofthe peptides are not substituted. In some embodiments, one or more(e.g., one, two, three, four, or all five) of positions three, four,five, six, seven, and eight of the peptides are identical to a sequenceselected from SEQ ID NOs: 1-17. As used herein, the term position refersto a position starting from the N-terminal of a peptide. For example,position 3 of the peptide refers to the third amino acid residuestarting from the N-terminal of the peptide. In some embodiments, theresidues at positions three, four, five, six, seven, and eight of SEQ IDNOs: 1-17 contribute the most to the recognition of the peptide.

The disclosure further provides an amino acid sequence or a nucleotidesequence comprising, consisting of, or consisting essentially of, asequence that is at least 50% (e.g., 60%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%) identical to any sequence as described in this disclosure, e.g.,SEQ ID NOs: 1-17, SEQ ID NOs: 18 and 19, and a nucleotide sequenceencoding SEQ ID NOs: 1-17.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes).The length of a reference sequence aligned for comparison purposes is atleast 80% of the length of the reference sequence, and in someembodiments is at least 90%, 95%, or 100%. The amino acid residues ornucleotides at corresponding amino acid positions or nucleotidepositions are then compared. When a position in the first sequence isoccupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. For purposes of the presentdisclosure, the comparison of sequences and determination of percentidentity between two sequences can be accomplished using a Blosum 62scoring matrix with a gap penalty of 12, a gap extend penalty of 4, anda frameshift gap penalty of 5.

Also provided herein are peptides comprising or consisting of a firstamino acid sequence; and a second amino acid sequence that isheterologous to the first amino acid sequence. An amino acid sequencethat is “heterologous” to a first amino acid sequence, or the term“heterologous amino acid sequence,” is an amino acid sequence flankingthe first amino acid sequence, wherein the flanking sequence does notoccur in nature (e.g., the flanking sequence is not linked to the firstamino acid sequence in nature). The first amino acid sequence cancomprise, consist essentially of, or consist of any sequence asdescribed herein, e.g., SEQ ID NOs: 1-17, or any sequence derived fromSEQ ID NOs: 1-17 (e.g., a sequence with no more than four substitutionsof SEQ ID NOs: 1-17). The peptide with heterologous flanking amino acidsequence generally do not (and are selected such that do not) adverselyaffect the generation in the cell of an immunogenic peptide of SEQ IDNO: 1-17. The cellular machinery is expected to remove any additionalsequences in the peptide to yield an immunogenic peptide of SEQ ID NO:1-17, which peptide is presented by a class I or class II MHC moleculeto stimulate an immune response against BCMA or TACI-expressing cancercells.

A heterologous flanking sequence can be, for example, a sequence usedfor purification of the recombinant protein (e.g., FLAG, polyhistidine(e.g., hexahistidine), hemagglutinin (HA), glutathione-S-transferase(GST), or maltose-binding protein (MBP)). Heterologous sequences canalso be proteins useful as diagnostic or detectable markers, forexample, luciferase, green fluorescent protein (GFP), or chloramphenicolacetyl transferase (CAT). In some embodiments, the peptides can containall or part of an immunoglobulin molecule (e.g., all or part of animmunoglobulin heavy chain constant region).

In some embodiments, the heterologous sequence can comprise atherapeutic or immune-stimulating polypeptide sequence (e.g., a T helperepitope (e.g., a PADRE epitope or a Tetanus Toxoid universal T helpercell epitope) or all or part of a cytokine or chemokine) and/or acarrier (e.g., KLH) useful, e.g., in eliciting an immune response (e.g.,for antibody generation). In some embodiments, the peptide can containone or more linker peptide sequences. The peptide can also contain atargeting polypeptide. Heterologous sequences can be of varying lengthand in some cases can be longer sequences than the first amino acidsequences to which the heterologous amino acid sequences are attached.It is understood that a peptide containing a first amino acid sequenceand a second amino acid sequence that is heterologous to the first doesnot correspond in sequence to a naturally occurring protein.

Targeting polypeptides, as used herein, are polypeptides that target themoiety (or moieties) they are attached to (e.g., the first amino acidsequence) to specific tissues (e.g., to a lymph node) or cells (e.g., toan antigen presenting cell or other immune cell), or where in vitro,specific isolated molecules or molecular complexes. Targetingpolypeptides can be, e.g., an antibody (immunoglobulin) or antigenbinding fragment thereof or a ligand for a cell surface receptor. Anantibody (or antigen-binding fragment thereof) can be, e.g., amonoclonal antibody, a polyclonal antibody, a humanized antibody, afully human antibody, a single chain antibody, a chimeric antibody, oran Fab fragment, an F(ab′)2 fragment, an Fab′ fragment, an Fv fragment,or an scFv fragment of an antibody. Antibody fragments that include, orare, Fc regions (with or without antigen-binding regions) can also beused to target the reagents to Fc receptor-expressing cells (e.g.,antigen presenting cells such as interdigitating dendritic cells,macrophages, monocytes, or B cells). A ligand for a cell surfacereceptor can be, e.g., a chemokine, a cytokine (e.g., interleukins 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16), or a death receptorligand (e.g., FasL or TNFα).

In some embodiments, the heterologous sequence can comprise, e.g., a“transportation sequence” that aids in the delivery of the peptide tothe cell or to a specific compartment of a cell (e.g., the endoplasmicreticulum or Golgi apparatus). Transportation sequences can include,e.g., membrane translocating sequence, a transportan sequence, anantennapedia sequence, a cyclic integrin-binding peptide, and aTat-mediated peptide, or modified versions thereof.

A linker peptide can connect the first amino acid sequence to one ormore heterologous amino acid sequences. For example, a linker peptidecan connect the first amino acid sequence to a second amino acidsequence. In certain embodiments, a linker peptide can link/connect apeptide of any one of SEQ ID NOs: 1-17 with a second peptide selectedfrom SEQ ID NOs: 1-17. The linker peptide can, or contain, e.g.,stretches of amino acids where at least four to six amino acids areglycine. (See, e.g., Mancebo et al. (1990) Mol. Cell. Biol. 10:2492-2502). A linker can also be, or contain, six or more (e.g., seven,eight, nine, ten, eleven, or twelve or more) histidine residues. Thelinker peptide can contain, or be, at least one (e.g., one, two, three,four, five, six, seven, or eight or more) protease cleavage site(s). Theprotease sites can be, e.g., a trypsin, a chymotrypsin, or a factor Xacleavage site. Such protease sites can be useful, e.g., to separate afirst amino acid sequence from a heterologous sequence. For example,after expression and purification of a peptide containing a first aminoacid sequence joined to a polyhistidine sequence (e.g., forpurification) by a trypsin protease cleavage site, the polyhistidinesequence can be removed from first amino acid sequence by contacting thepeptide with trypsin.

In some embodiments, the disclosure provides a peptide (e.g., any one ofSEQ ID NOs: 1-17) that can have at the amino-terminal end and/orcarboxy-terminal end up to 200 (e.g., one, two, three, four, five, six,seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200) aminoacids that are heterologous or are present in the native protein.

In some embodiments, the peptide can include a sequence that is at least40%, at least 50%, at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any sequence as described herein (e.g., any one of SEQ IDNOs: 1-17). In some embodiments, the sequence can have equal to or atleast one, two, three, four, five, six, seven, eight, nine, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 amino acids selected from any sequenceas described herein (e.g., any one of SEQ ID NOs: 1-17). In someembodiments, the sequence is SEQ ID NO: 13. In some embodiments, thesequence is SEQ ID NO: 14. In some embodiments, the sequence is SEQ IDNO: 16.

In some embodiments, the peptide can have an additional sequence. Theadditional sequence can be located at the amino-terminal end or thecarboxy-terminal of the peptide. In some embodiments, the additionalsequence can have at least one, two, three, four, five, six, seven,eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acids. In someembodiments, the additional sequence can have up to one, two, three,four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or200 amino acids.

In certain instances, the disclosure encompasses any combination of 2 ormore peptides selected from SEQ ID NOs: 13-17.

The peptides described herein can bind to a major histocompatibilitycomplex (MHC) molecule (e.g., an MHC class I molecule or an MHC class IImolecule). In humans, the MEW is known as the HLA complex. An “HLAsupertype or family,” as used herein, refers to sets of HLA moleculesgrouped on the basis of shared peptide-binding specificities. HLA classI molecules that share somewhat similar binding affinity for peptidesbearing certain amino acid motifs are grouped into HLA supertypes. Theterms HLA superfamily, HLA supertype family, HLA family, and HLA xx-likemolecules (where xx denotes a particular HLA type), are synonyms. Typesof HLA class I molecules include, e.g., HLA-A1, HLA-A2, HLA-A3, HLA-A24,HLA-B7, HLA-B27, HLA-B44, HLA-B58, or HLA-B62. Such HLA molecules aredescribed in detail in U.S. Pat. No. 7,026,443, the entire disclosure ofwhich is incorporated by reference in its entirety.

A peptide can bind to an MHC molecule with high affinity or intermediateaffinity. As used herein, “high affinity” binding of a peptide to an HLAclass I molecule is defined as a binding with a dissociation constant(KD) of less than 50 (e.g., 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.5,0.1, or less than 0.05) nM. “Intermediate affinity” is a binding of apeptide to an HLA class I molecule with a KD of between about 50 nM andabout 500 nM (e.g., 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 115,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 nM). “Highaffinity” binding of a peptide to HLA class II molecules is defined asbinding with a KD of less than 100 (e.g., 95, 90, 85, 80, 75, 70, 65,60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, 0.5, 0.1, or less than0.05) nM. “Intermediate affinity” of a peptide for an HLA class IImolecule is binding with a KD of between about 100 and about 1000 nM(e.g., 100, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490,500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630,640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770,780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910,920, 930, 940, 950, 960, 970, 980, 990, or 1000 nM). Methods fordetermining the binding affinity of a peptide and an MHC molecule areknown in the art. Suitable methods are also described in, e.g., U.S.Pat. No. 7,026,443.

The peptides described herein can also be, in association with an MHCmolecule, recognized by an antigen specific T cell receptor on a T cell.A variety of suitable methods can be used to determine whether apeptide, in association with an MHC molecule, is recognized by a T cellreceptor on a T cell. For example, peripheral blood lymphocytes (PBL)from normal subjects can be cultured with a test peptide in the presenceof antigen presenting cells in vitro over a period of several weeks. Tcells specific for the peptide become activated during this time and canbe detected using, e.g., proliferation assays (carboxyfluorsceinsuccinimidyl ester (CFSE) assays of H-thymidine assays), limitingdilution assays, cytotoxicity assays (e.g., calcein-release assays), orcytokine- (e.g., IFNγ), lymphokine-, or 51Cr-release assays (see, e.g.,Wentworth, P. A. et al., Mol. Immunol. 32: 603, 1995; Celis, E. et al,Proc. Natl. Acad. Sci. USA 91: 2105, 1994; Tsai, V. et al., J. Immunol.158: 1796, 1997; Kawashima, I. et al., Human Immunol. 59: 1, 1998, thedisclosures of each of which are incorporated by reference in theirentirety). A suitable in vivo method involves immunizing HLA transgenicmice, wherein peptides in adjuvant are administered subcutaneously toHLA transgenic mice and several weeks following immunization,splenocytes are removed and cultured in vitro in the presence of testpeptide for approximately one week, peptide-specific T cells aredetected using, e.g., a 51Cr-release assay (see, e.g., Wentworth, P. A.et al., J. Immunol. 26: 97, 1996; Wentworth, P. A. et al., Int. Immunol.8: 651, 1996; Alexander, J. et al., J. Immunol. 159: 4753, 1997, thedisclosures of each of which are incorporated by reference in theirentirety).

Additionally, direct quantification of antigen-specific T cells can beperformed by staining T cells with detectably-labeled MHC complexes suchas any of the MHC molecule multimer compositions described herein orHLA-I tetramers (e.g., as described in Altman, J. D. et al., Proc. Natl.Acad. Sci. USA 90: 10330, 1993 and Altman, J. D. et al., Science 274:94, 1996, the disclosures of each of which are incorporated by referencein their entirety).

In some embodiments, the peptides can be further modified (e.g., aminoacids of the peptides can be substituted) in order to modulate (e.g.,increase or decrease) one of more properties of the peptides. Forexample, one or more (e.g., two, three, or four) amino acids of one ofthe peptides described herein can be substituted in order to increasethe affinity of the peptide for an MHC molecule. In some embodiments, anamino acid of one of the peptides described herein (e.g., a T cellReceptor contacting amino acid residue of the peptide) can be modifiedin order to enhance a binding interaction between a T cell receptor andthe peptide (in the context of an MHC molecule). Such modified peptidesare often referred to as “altered peptide ligands.” (See, e.g., Kalergiset al. (2000) J Immunol. 165(1): 280; Conlon et al. (2002) Science 1801;and International Publication No. WO02070003, the disclosure of each ofwhich is incorporated by reference in their entirety). Suitable methodsfor modifying the peptides as well as determining the effect of themodification are described in, e.g., Collins et al. (ImmunologicalReviews (1998) 163: 151-160, the disclosure of which is incorporated byreference in its entirety).

The disclosure further provides a composition comprising any peptides asdescribed herein. Furthermore, to counter the tumor's ability to evadetherapies directed against it, the composition can comprise a variety ofspecific peptides to induce the immune response. For example, more thanone epitope from the same protein can be included in the composition,e.g., the composition can contain at least one, at least two, at leastthree, at least four, at least five, at least six, or at least sevendifferent peptides derived from BCMA or TACI. In addition, combinationsor mixtures of at least one (e.g., at least two, three, four, five)peptides derived from BCMA and at least one (e.g., at least two, three,four, five) peptides derived from TACI can also be used. Thus, in someembodiments, the disclosure provides a composition comprising at least2, 3, 4, 5, 6, 7, or 8 BCMA-derived peptides (e.g., at least twoBCMA-derived peptides selected from, e.g., SEQ ID NOs: 1-6 and 13-14).In some embodiments, the disclosure provides a composition comprising atleast 2, 3, 4, 5, 6, 7, or 8 TACI-derived peptides (e.g., at least twoTACI-derived peptides selected from e.g., SEQ ID NOs: 7-12 and 16-17).In some embodiments, the disclosure provides a composition comprising atleast one (e.g., at least 2, 3, 4, 5, 6, 7, or 8) BCMA-derived peptidesand at least one (e.g., at least 2, 3, 4, 5, 6, 7, or 8) TACI-derivedpeptides.

In some embodiments, the composition further comprises an immunestimulatory agent (e.g., a cytokine or a T helper epitope). The T helperepitope can be a PADRE sequence or a universal Tetanus Toxoid T helper(TT Th) epitope. In some embodiments, the composition comprises anadjuvant, such as Freund's complete adjuvant, Freund's incompleteadjuvant, alum, a ligand for a Toll receptor, QS21, RIBI, or similarimmunostimulatory agent. Adjuvants also include, e.g., cholera toxin(CT), E. coli heat labile toxin (LT), mutant CT (MCT) (Yamamoto et al.(1997) J. Exp. Med. 185: 1203-1210) and mutant E. coli heat labile toxin(MLT) (Di Tommaso et al. (1996) Infect. Immunity 64: 974-979). In someembodiments, the composition comprises a toll like receptor-3 ligand(e.g., Poly ICLC), interferon alfa (IFNα), interferon gamma (IFNγ), orGranulocyte-macrophage colony-stimulating factor (GM-CSF). In someembodiments, the composition further comprises an immune agonist, e.g.,an anti-OX40 antibody or an anti-GITR antibody.

In some embodiments, the composition comprises a chemotherapeutic agent(e.g., Lenalidomide). In some embodiments, the composition comprises ahistone deacetylase 6 (HDAC6) inhibitor (e.g., ACY241; See Niesvizky, etal. “ACY-241, a novel, HDAC6 selective inhibitor: synergy withimmunomodulatory (IMiD®) drugs in multiple myeloma (MM) cells and earlyclinical results (ACE-MM-200 Study).” (2015): 3040-3040; Bae et al.“Histone deacetylase (HDAC) inhibitor ACY241 enhances anti-tumoractivities of antigen-specific central memory cytotoxic T lymphocytesagainst multiple myeloma and solid tumors.” Leukemia (2018): 1). In someembodiments, the composition comprises a checkpoint inhibitor (e.g.,anti-LAG3 antibody) or an immune agonist (e.g., anti-OX40). In someembodiments, the composition comprises an antibody (e.g., humanantibody) the specifically binds to PD-1, CTLA-4, LAG-3, BTLA, PD-L1,CD27, CD28, CD40, CD47, 4-1BB (CD137), CD154, TIGIT, TIM-3, GITR(CD357), OX40, CD20, EGFR, or CD319.

In some embodiments, the composition comprises a peptide comprising asequence set forth in SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 16,and Lenalidomide. In some embodiments, the composition comprises apeptide comprising a sequence set forth in SEQ ID NO: 13, SEQ ID NO: 14,or SEQ ID NO: 16, and an anti-OX40 antibody, an anti-LAG3 antibody,and/or an anti-GITR antibody.

Nucleic Acids and Methods for Producing the Peptides

The disclosure also features nucleic acid sequences (as well as nucleicacid vectors containing nucleic acid sequences) encoding, and methodsfor producing, one or more (e.g., one, two, three, four, five, six,seven, eight, nine, 10, 11, 12, 13, or 14) of any of the peptidesdescribed herein. Such methods can include the steps of: optionally,providing a cell (or group of cells) comprising a nucleic acid vectorcontaining a nucleic acid sequence encoding one of more of any of thepeptides described herein, the nucleic acid sequence being operablylinked to an expression control sequence, and culturing the cell underconditions that permit the expression of the peptides. The methods canalso include the step of isolating the one or more peptides from thecell, or from the medium in which the cell was cultured. Thus, in oneaspect, the disclosure provides RNA-based therapeutics and DNA-basedtherapeutics including e.g., cancer vaccines. In some embodiments, thecancer vaccines can have a polynucleotide as described herein (e.g., apolynucleotide encoding SEQ ID NOS: 1-17). In some instances, thepolynucleotide encodes a peptide that is identical to one of SEQ IDNOs.: 1-17, except having 1 to 4 amino acid substitutions. In somecases, the substitution is at one or more of positions 1, 2, or 9. Insome cases, the peptide is 2 to 30 amino acids in length. In some cases,the nucleic acid can include regulatory sequences (e.g., start codon,stop, codon polyA tail). In some embodiments, the RNA/DNA cancervaccines can be formulated within a polymeric or liposomal nanocarrier(e.g., a nanoparticle).

Suitable methods for constructing nucleic acid sequences and vectors(e.g., expression vectors) for recombinant expression of one or more ofthe peptides described herein are well known to those skilled in the artand described in, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual Second Edition vol. 1, 2 and 3. Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, N.Y., USA, November 1989, the disclosure ofwhich is incorporated by reference in its entirety. The nucleic acidsand vectors can be used, e.g., to express the peptides in a wide varietyof host cells including, e.g., a bacterial, a yeast, or a mammaliancell. The nucleic acids and vectors can also be used in, e.g., in vivoand ex vivo methods as described below. The peptide-coding sequences canbe operably-linked to a promoter, a regulatory element, or an expressioncontrol sequence. The promoter and/or enhancer elements can direct theexpression of the peptides encoded by the nucleic acids. Enhancersprovide expression specificity in terms of time, location, and level.Unlike a promoter, an enhancer can function when located at variabledistances from the transcription initiation site, provided a promoter ispresent. An enhancer can also be located downstream of the transcriptioninitiation site or in an exon of the relevant gene. To bring a codingsequence under the control of a promoter, it is necessary to positionthe translation initiation site of the translational reading frame ofthe peptide between one and about fifty nucleotides downstream (3′) ofthe promoter. Promoters of interest include, but are not limited to, thecytomegalovirus hCMV immediate early gene, the early or late promotersof SV40 adenovirus, the lac system, the trp system, the TAC system, theTRC system, the major operator and promoter regions of phage A, thecontrol regions of fd coat protein, the promoter for 3 phosphoglyceratekinase, the promoters of acid phosphatase, and the promoters of theyeast a mating factors, the adenoviral EIb minimal promoter, or thethymidine kinase minimal promoter.

The peptide-coding sequences, or vectors containing the peptide-codingsequences, can contain a leader sequence that encodes a signal peptide.The leader sequence can be at the 5′ end of the sequence encoding one ormore of the peptides described herein. The signal peptide can beimmediately N-terminal of a given peptides or can be separated from itby one or more (e.g., 2, 3, 4, 6, 8, 10, 15 or 20) amino acids, providedthat the leader sequence is in frame with the nucleic acid sequenceencoding the peptides. The signal peptide, which is generally cleavedfrom the peptide prior to secretion (unless of course the signal peptidedirects the insertion of a transmembrane protein), directs the peptideto which it is attached into the lumen of the host cell endoplasmicreticulum (ER) during translation and the peptides are then secreted,via secretory vesicles, into the environment of the host cell. Usefulsignal peptides include, e.g., native leader sequences of cytokines orgrowth factors, KDEL (Lys-Asp-Glu-Leu), or any signal sequencesdescribed in, e.g., U.S. Pat. No. 5,827,516, the disclosure of which isincorporated herein by reference in its entirety.

In some embodiments, the 5′ end of a peptide-coding sequence can includea non-native ATG “start sequence.” That is, e.g., an ATG sequence can beadded to a nucleic acid encoding a peptide to ensure that the peptide isproperly transcribed and translated. Although a leader sequencegenerally includes an ATG start sequence, in embodiments where it doesnot, the ATG sequence can be added at the 5′ end of a nucleic acidencoding the leader sequence.

Suitable methods for constructing peptide-coding sequences andexpression vectors are well known to those skilled in the art anddescribed in, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual Second Edition vol. 1, 2 and 3. Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, N.Y., USA, November 1989; the disclosure ofwhich is incorporated herein by reference in its entirety.

A recombinant vector can be introduced into a cell using a variety ofmethods, which methods can depend, at least in part, on the type of cellinto which the nucleic acid is introduced. For example, bacterial cellscan be transformed using methods such as electroporation or heat shock.Methods for transfecting yeast cells include, e.g., the spheroplasttechnique or the whole-cell lithium chloride yeast transformation method(see, e.g., U.S. Pat. No. 4,929,555; Hinnen et al. (1978) Proc. Nat.Acad. Sci. USA 75: 1929; Ito et al. (1983) J. Bacteriol. 153: 163; U.S.Pat. No. 4,879,231; and Sreekrishna et al. (1987) Gene 59: 115, thedisclosures of each of which are incorporated herein by reference intheir entirety). Transfection of animal cells can feature, for example,the introduction of a vector to the cells using calcium phosphate,electroporation, heat shock, liposomes, or transfection reagents such asFUGENE® or LIPOFECTAMINE®, or by contacting naked nucleic acid vectorswith the cells in solution (see, e.g., Sambrook et al., supra).

Expression systems that can be used for small or large scale productionof the peptides described herein include, but are not limited to,microorganisms such as bacteria (for example, E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmidDNA expression vectors; fungus (e.g., yeast (for example, Saccharomycesand Pichia)) transformed with recombinant yeast expression vectors;insect cell systems infected with recombinant virus expression vectors(for example, baculovirus); plant cell systems infected with recombinantvirus expression vectors (for example, cauliflower mosaic virus (CaMV)and tobacco mosaic virus (TMV)) or transformed with recombinant plasmidexpression vectors (for example, Ti plasmid); or mammalian cell systems(for example, COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, and NIH 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (for example, themetallothionein promoter) or from mammalian viruses (for example, theadenovirus late promoter, a CMV promoter, an SV40 promoter, or thevaccinia virus 7.5K promoter). Also useful as host cells are primary orsecondary cells obtained directly from a mammal, transfected with aplasmid vector or infected with a viral vector (e.g., viral vectors suchas herpes viruses, retroviruses, vaccinia viruses, attenuated vacciniaviruses, canary pox viruses, adenoviruses and adeno-associated viruses,among others).

Following the expression of any of the peptides described herein, thepeptides can be isolated from the cultured cells, or from the media inwhich the cells were cultured, using standard techniques. Methods ofisolating proteins are known in the art and include, e.g., liquidchromatography (e.g., HPLC), affinity chromatography (e.g., metalchelation or immunoaffinity chromatography), ion-exchangechromatography, hydrophobic-interaction chromatography, precipitation,or differential solubilization.

Smaller peptides (e.g., peptides having less than 200 (e.g., less than175, less than 150, less than 125, less than 100, less than 90, lessthan 80, less than 70, or less than 60) amino acids) can be chemicallysynthesized by standard chemical means such as FMOC solid-phasesynthesis.

The peptides described herein can, but need not, be isolated. The term“isolated,” as applied to any of the peptides described herein, refersto a peptide, a fragment thereof, (or for compositions, a macromolecularcomplex), that has been separated or purified from components (e.g.,proteins or other naturally-occurring biological or organic molecules)which naturally accompany it. It is understood that recombinantmolecules (e.g., recombinant peptides) will always be “isolated.”Typically, a peptide (or fragment or macromolecular complex) is isolatedwhen it constitutes at least 60%, 70%, 80%, or 90% by weight, of thetotal molecules of the same type in a preparation, e.g., at least 60%,70%, 80%, or 90% of the total molecules of the same type in a sample.For example, a peptide described herein is considered isolated when itconstitutes at least 60%, 70%, 80%, or 90% by weight, of the totalprotein in a preparation or sample. In some embodiments, a molecule inthe preparation consists of at least 75%, at least 90%, or at least 99%,by weight, of the total molecules of the same type in a preparation.

Similarly, the peptide-coding sequences or vectors containing thepeptide-coding sequences described herein can also be isolated. The term“isolated,” as applied to any of the peptide-coding sequences or vectorsdescribed herein, refers to a peptide-coding sequence or vector, afragment thereof that has been separated or purified from components(e.g., nucleic acids, proteins, or other naturally-occurring biologicalor organic molecules) which naturally accompany it. It is understoodthat recombinant molecules (e.g., recombinant vectors or peptide-codingsequences) will always be “isolated.” Typically, a peptide-codingsequence or vector (or fragment thereof) is isolated when it constitutesat least 60%, 70%, 80%, or 90% by weight, of the total molecules of thesame type in a preparation, e.g., at least 60%, 70%, 80%, or 90% of thetotal molecules of the same type in a sample. For example, apeptide-coding sequence or vector described herein is consideredisolated when it constitutes at least 60%, 70%, 80%, or 90% by weight,of the total nucleic acid in a preparation or sample. In someembodiments, a molecule in the preparation consists of at least 75%, atleast 90%, or at least 99%, by weight, of the total molecules of thesame type in a preparation.

In some embodiments, the isolated peptides, peptide-coding sequences, orvectors can be frozen, lyophilized, or immobilized and stored underappropriate conditions, which allow the molecules to retain activity(e.g., the ability of a peptide to bind to an WIC molecule such as anWIC class I molecule or an WIC class II molecule, or the ability of avector to support expression of a peptide in a cell).

Processing of the Peptides

Following the expression or synthesis of any of the peptides describedherein, the peptides can be further processed. The further processingcan include chemical or enzymatic modifications to peptides or, in caseswhere the peptides are modified, the processing can include enzymatic orchemical alterations of existing modifications, or both. The additionalprocessing of the peptides can include the addition (covalent ornon-covalent joining) of a heterologous amino acid sequence such as, butnot limited to, any of the heterologous amino acid sequences describedherein. Enzymatic treatment can involve contacting a peptide with, e.g.,one or more proteases, phosphatases, or kinases under conditions thatallow the peptide to be modified. Enzymatic treatment can involvecontacting a peptide with one or more enzymes (e.g., anoligosaccharyltransferase or a mannosidase) capable of glycosylating, ormodifying the glycosylation of, the peptide.

The processing can include the addition of, e.g., a detectable label toa peptide. For example, a peptide can be detectably labeled with anenzyme (e.g., horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase), a fluorescent material (e.g.,umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine, fluorescein, dansyl chloride, allophycocyanin(APC), or phycoerythrin), a luminescent material (e.g., a lanthanide orchelate thereof), a bioluminescent material (e.g., luciferase,luciferin, or aequorin), or a radionuclide (e.g., ³H, ³²P, ³³P, ¹²⁵I or³⁵S).

The processing can also involve the coupling of the peptide to a polymer(e.g., a polyalkylene glycol moiety such as a polyethylene glycolmoiety), or a nanoparticle. In some embodiments, the polymer is coupledto the polypeptide at a site on the peptide that is an N terminus. Insome embodiments, a peptide can contain one or more internal amino acidinsertions that provide an internal polymer conjugation site to which apolymer can be conjugated.

Methods for Inducing an Immune Response

The disclosure also provides a variety of methods for inducing an immuneresponse in a subject (e.g., peptide based vaccination,nanoparticle-based immunotherapy, APC-based immunotherapy, T cell-basedimmunotherapy, CAR T cell-based immunotherapy, or induced pluripotentstem cell-approaches). The methods for inducing an immune response in asubject can include, e.g., the step of administering to a subject one ormore of the compositions described herein (e.g., any of peptides (orexpression vectors containing nucleic acid sequences encoding thepeptides) described herein (or any of the pharmaceutical compositionscontaining one or more peptides (or vectors) described herein)). In someembodiments, the composition described herein is used as a vaccine.

Any of the peptides described herein can be used to stimulate an immuneresponse by use of a nucleic acid expression system that encodes one ormore of the peptides described herein. That is, methods for inducing animmune response in a subject can include the step of administering to asubject an expression vector containing nucleic acid sequences encodingone or more of the peptides described herein (or a pharmaceuticalcomposition containing the expression vector). The immune response canbe a CD8+ T cell, a CD4+ T cell, a cytotoxic T lymphocyte (CTL), a TH1response, a TH2 response, or a combination of both types of responses.

Any of the above methods can also be, e.g., methods for treating orpreventing (prophylaxis against) a cancer (e.g., plasma cell disordersuch as multiple myeloma, or any other cancer expressing BCMA and/orTACI) in a subject. When the terms “prevent,” “preventing,” or“prevention” are used herein in connection with a given treatment for agiven condition, they mean that the treated subject either does notdevelop a clinically observable level of the condition at all (e.g., thesubject does not exhibit one or more symptoms of the condition or, inthe case of a cancer, the subject does not develop a detectable level ofthe cancer), or the condition develops more slowly and/or to a lesserdegree (e.g., fewer symptoms or lower numbers of cancer cells in thesubject) in the subject than it would have absent the treatment. Theseterms are not limited solely to a situation in which the subjectexperiences no aspect of the condition whatsoever. For example, atreatment will be said to have “prevented” the condition if it is givenduring, e.g., during an early diagnosis of a cancer (e.g., the detectionof a few cancer cells in a sample from the subject) that would have beenexpected to produce a given manifestation of the condition (an advancedcancer), and results in the subject's experiencing fewer and/or mildersymptoms of the condition than otherwise expected. A treatment can“prevent” a cancer (e.g., a plasma cell disorder such as multiplemyeloma) when the subject displays only mild overt symptoms of thecancer. “Prevention” does not imply that there must have been nodevelopment of even a single cancer cell by a subject so treated.

Generally, a peptide delivered to the subject will be suspended in apharmaceutically-acceptable carrier (e.g., physiological saline) andadministered orally, rectally, or parenterally, e.g., injectedintravenously, subcutaneously, intramuscularly, intrathecally,intraperitoneally, intrarectally, intravaginally, intranasally,intragastrically, intratracheally, or intrapulmonarily.

Administration can be by periodic injections of a bolus of thepharmaceutical composition or can be uninterrupted or continuous byintravenous or intraperitoneal administration from a reservoir which isexternal (e.g., an IV bag) or internal (e.g., a bioerodable implant, abioartificial organ, or a colony of implanted reagent production cells).See, e.g., U.S. Pat. Nos. 4,407,957, 5,798,113 and 5,800,828, eachincorporated herein by reference in its entirety.

Conventional and pharmaceutically acceptable routes of administration ofa therapeutic nucleic acid include, but are not necessarily limited to,intramuscular, subcutaneous, intradermal, transdermal, intravenous,rectal (e.g., enema, suppository), oral, intragastric, intranasal andother routes of effective inhalation routes, and other parenteral routesof administration. Routes of administration may be combined, if desired,or adjusted depending upon the nucleic acid molecule and/or the desiredeffect on the immune response. Methods for administering a nucleic acidto a subject can include a variety of well-known techniques such asvector-mediated gene transfer (e.g., viral infection/transfection, orvarious other protein-based or lipid-based gene delivery complexes) aswell as techniques facilitating the delivery of “naked” polynucleotides(such as electroporation, “gene gun” delivery, and various othertechniques used for the introduction of polynucleotides to a subject ora cell of a subject).

In general, the dosage of a peptide or a nucleic acid required dependson the choice of the route of administration; the nature of theformulation; the nature or severity of the subject's illness; the immunestatus of the subject; the subject's size, weight, surface area, age,and sex; other drugs being administered; and the judgment of theattending medical professional.

Suitable dosages of peptide for inducing an immune response are in therange of 0.000001 to 10 mg of the reagent or antigenic/immunogeniccomposition per kg of the subject. Wide variations in the needed dosageare to be expected in view of the variety of reagents and the differingefficiencies of various routes of administration. For example, nasal orrectal administration may require higher dosages than administration byintravenous injection. Variations in these dosage levels can be adjustedusing standard empirical routines for optimization as is well understoodin the art. Administrations can be single or multiple (e.g., 2-, 3-, 4-,6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold). For example, a peptidecan be administered as an initial immunization and then administered oneor more times subsequently as a booster immunization.

The dose of nucleic acid administrated to a subject, in the context ofthe methods described herein, should be sufficient to effect abeneficial therapeutic response in the subject over time, or toalleviate symptoms. Although the dosage used will vary depending on,e.g., the subject or the clinical goals to be achieved. A suitabledosage range is one which provides up to about 1 μg, to about 1,000 μg,to about 5,000 μg, to about 10,000 μg, to about 25,000 μg or about50,000 μg of nucleic acid per ml of carrier in a single dosage.

In order to optimize therapeutic efficacy (e.g., the efficacy of the oneor more peptides or the nucleic acids encoding the peptides to induce animmune response in a subject), compositions containing the peptides ornucleic acids can be first administered at different dosing regimens.The unit dose and regimen depend on factors that include, e.g., thespecies of mammal, its immune status, the body weight of the mammal. Thefrequency of dosing for a pharmaceutical composition (e.g., apharmaceutical composition containing one or more peptides or one ormore nucleic acid sequences encoding one or more of the peptidesdescribed herein) is within the skills and clinical judgement of medicalpractitioners (e.g., doctors or nurses). Typically, the administrationregime is established by clinical trials which may establish optimaladministration parameters. However, the practitioner may vary suchadministration regimes according to the subject's age, health, weight,sex and medical status.

In some embodiments, a pharmaceutical composition can be administered toa subject at least two (e.g., three, four, five, six, seven, eight,nine, 10, 11, 12, 15, or 20 or more) times. For example, apharmaceutical composition can be administered to a subject once a monthfor three months; once a week for a month; once a year for three years,once a year for five years; once every five years; once every ten years;or once every three years for a lifetime.

As defined herein, a “therapeutically effective amount” of a peptide ora nucleic acid encoding a peptide is an amount of the peptide or nucleicacid that is capable of producing an immune response in a treatedsubject. A therapeutically effective amount of a peptide (i.e., aneffective dosage) includes milligram, microgram, nanogram, or picogramamounts of the agent per kilogram of subject or sample weight (e.g.,about 1 nanogram per kilogram to about 500 micrograms per kilogram,about 1 microgram per kilogram to about 500 milligrams per kilogram,about 100 micrograms per kilogram to about 5 milligrams per kilogram, orabout 1 microgram per kilogram to about 50 micrograms per kilogram). Atherapeutically effective amount of a nucleic acid also includesmicrogram, nanogram, or picogram amounts of the agent per kilogram ofsubject or sample weight (e.g., about 1 nanogram per kilogram to about500 micrograms per kilogram, about 1 microgram per kilogram to about 500micrograms per kilogram, about 100 micrograms per kilogram to about 500micrograms per kilogram, or about 1 microgram per kilogram to about 50micrograms per kilogram).

As defined herein, a “prophylactically effective amount” of a peptide ornucleic acid encoding a peptide is an amount of the peptide or nucleicacid that is capable of producing an immune response against a cancercell (e.g., a multiple myeloma) in a treated subject, which immuneresponse is capable of preventing the development of a cancer in asubject or is able to substantially reduce the chance of a subjectdeveloping or continue developing a cancer. A prophylactically effectiveamount of a peptide (i.e., an effective dosage) includes milligram,microgram, nanogram, or picogram amounts of the agent per kilogram ofsubject or sample weight (e.g., about 1 nanogram per kilogram to about500 micrograms per kilogram, about 1 microgram per kilogram to about 500milligrams per kilogram, about 100 micrograms per kilogram to about 5milligrams per kilogram, or about 1 microgram per kilogram to about 50micrograms per kilogram). A prophylactically effective amount of anucleic acid also includes microgram, nanogram, or picogram amounts ofthe agent per kilogram of subject or sample weight (e.g., about 1nanogram per kilogram to about 500 micrograms per kilogram, about 1microgram per kilogram to about 500 micrograms per kilogram, about 100micrograms per kilogram to about 500 micrograms per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram).

In some embodiments, the methods can also include determining if animmune response occurred in a subject after administering thecompositions to the subject. Suitable methods for determining whether animmune response occurred in a subject include use of immunoassays todetect, e.g., the presence of antibodies specific for a peptide in abiological sample from the subject. For example, after theadministration of the peptide to the subject, a biological sample (e.g.,a blood sample) can be obtained from the subject and tested for thepresence of antibodies specific for the peptide(s). An immune responsecan also be detected by assaying for the presence or amount of activatedT cells in a sample. Such assays include, e.g., proliferation assays,limiting dilution assays, cytotoxicity assays (e.g., lymphokine- or51Cr-release assays).

In some embodiments, the methods described herein (e.g., therapeuticsthat increase the BCMA and TACI-specific responses, vaccines, celltherapies, antibodies, and/or therapeutic approach targeting sometargets other than BCMA and TACI) can also include administering to asubject various types of compounds to enhance the BCMA and TACI-specificresponses (e.g. cytokines and chemokines), checkpoint inhibitors (e.g.,an anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, and/or anti-TIM3antibody), immune agonists (e.g., an anti-OX40 or anti-GITR antibody),immune modulators (e.g., Lenalidomide, Pomalidomide, HDAC inhibitorssuch as ACY241), and/or adjuvants.

The methods can also include the step of administering to the subjectone or more chemotherapeutic agents, one or more forms of ionizingradiation, or one or more immunomodulatory agents. The one or more formsof ionizing radiation can be gamma-irradiation, X-irradiation, orbeta-irradiation. The one or more chemotherapeutic agents can beselected from the group consisting of cisplatin, carboplatin,procarbazine, mechlorethamine, cyclophosphamide, camptothecin,adriamycin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea,dactinomycin, daunorubicin, doxorubicin, bleomycin, lincomycin,mitomycin, etoposide, verapamil, podophyllotoxin, tamoxifen, taxol,thalidomide, lenalidomide, a proteasome inhibitor (e.g., bortezomib), anhsp90 inhibitor (e.g., tanespimycin), transplatinum, 5-fluorouracil,vincristine, vinblastine, methotrexate, or an analog of any of theaforementioned. Immunomodulatory agents include, e.g., a variety ofchemokines and cytokines such as Interleukin 2 (IL-2),granulocyte/macrophage-colony stimulating factor (GM-CSF), andInterleukin 12 (IL-12). In some embodiments, the peptide or a nucleicacid encoding the peptide can be administered with an immune modulatorsuch as a Toll Receptor ligand or an adjuvant.

In some embodiments, the additional therapeutic agent is a histonedeacetylase 6 (HDAC6) inhibitor (e.g., ACY241).

In some embodiments, the one or more additional therapeutic agents canbe an immunomodulatory agent, a checkpoint inhibitor (e.g., anti-LAG3antibody) or an immune agonist (e.g., anti-OX40 antibody, anti-GITRantibody).

In some embodiments, the additional therapeutic agent can comprise oneor more inhibitors selected from the group consisting of an inhibitor ofB-Raf, an EGFR inhibitor, an inhibitor of a MEK, an inhibitor of ERK, aninhibitor of K-Ras, an inhibitor of c-Met, an inhibitor of anaplasticlymphoma kinase (ALK), an inhibitor of a phosphatidylinositol 3-kinase(PI3K), an inhibitor of an Akt, an inhibitor of mTOR, a dual PI3K/mTORinhibitor, an inhibitor of Bruton's tyrosine kinase (BTK), and aninhibitor of Isocitrate dehydrogenase 1 (IDH1) and/or Isocitratedehydrogenase 2 (IDH2). In some embodiments, the additional therapeuticagent is an inhibitor of indoleamine 2,3-dioxygenase-1) (IDO1) (e.g.,epacadostat).

In some embodiments, the additional therapeutic agent can comprise oneor more inhibitors selected from the group consisting of an inhibitor ofHER3, an inhibitor of LSD1, an inhibitor of MDM2, an inhibitor of BCL2,an inhibitor of CHK1, an inhibitor of activated hedgehog signalingpathway, and an agent that selectively degrades the estrogen receptor.

In some embodiments, the combination therapy includes one or more of thefollowing:

(A) compounds that can enhance the BCMA and TACI-specific responses suchas (1) cytokines and chemokines, (2) checkpoint inhibitors includinge.g., an anti-PD1, anti-PDL1, anti-CTLA4, anti-LAG3, and/or anti-TIM3antibody, (2) immune agonists including e.g., an anti-OX40 and/oranti-GITR antibody, (3) immune modulators including e.g., Lenalidomide,Pomalidomide, HDAC inhibitors such as ACY241, (4) adjuvant;

(B) therapeutics which can increase the BCMA and TACI-specificresponses, including any therapeutics as described herein (e.g.,vaccine, cell therapies and/or antibodies) or independent approach thattarget other targets (e.g., non-BCMA or non-TACI-targeting therapy); and

(C) additional compounds such as one or more inhibitors selected fromthe group consisting of an inhibitor of B-Raf, an EGFR inhibitor, aninhibitor of a MEK, an inhibitor of ERK, an inhibitor of K-Ras, aninhibitor of c-Met, an inhibitor of anaplastic lymphoma kinase (ALK), aninhibitor of a phosphatidylinositol 3-kinase (PI3K), an inhibitor of anAkt, an inhibitor of mTOR, a dual PI3K/mTOR inhibitor, an inhibitor ofBruton's tyrosine kinase (BTK), and an inhibitor of Isocitratedehydrogenase 1 (IDH1) and Isocitrate dehydrogenase 2 (IDH2), and/orinhibitor of indoleamine 2,3-dioxygenase-1) (IDO1) (e.g., epacadostat).

In some embodiments, the additional therapeutic agent can comprise oneor more therapeutic agents selected from the group consisting ofTrabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib,Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin,Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib,Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, anHsp90 inhibitor, Ad-GM-CSF, Temozolomide, IL-2, IFNα, vinblastine,Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine,lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, andenzastaurin.

In some embodiments, the additional therapeutic agent can comprise oneor more therapeutic agents selected from the group consisting of anadjuvant, a TLR agonist, tumor necrosis factor (TNF) alpha, IL-1, HMGB1,an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an IL-17antagonist, an HVEM antagonist, an ICOS agonist, a treatment targetingCX3CL1, a treatment targeting CXCL9, a treatment targeting CXCL10, atreatment targeting CCL5, an LFA-1 agonist, an ICAM1 agonist, and aSelectin agonist.

In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin,pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to thesubject.

In some embodiments, the additional therapeutic agent is an antibody(e.g., human antibody) the specifically binds to PD-1, CTLA-4, LAG-3,BTLA, PD-L1, CD27, CD28, CD40, CD47, 4-1BB (CD137), CD154, TIGIT, TIM-3,GITR (CD357), OX40, CD20, EGFR, or CD319. In some embodiments, theadditional therapeutic agent is an anti-OX40 antibody, an anti-PD-L1antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGITantibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or ananti-GITR antibody.

The disclosure also provides a virus comprising nucleic acids encodingone or more peptides as described herein or a virus particle comprisingone or more peptides as described herein. Various viruses can be used,e.g., retrovirus, lentivirus, adenovirus, adeno-associated virus,alphavirus and the like. These viruses can be used to deliver thepeptide or nucleic acids encoding the peptides thereof to a subject toinduce immune response. Similarly, liposomes that comprise one or morepeptides as described herein and/or nucleic acids encoding the peptidesthereof can also be used to deliver the peptides or the nucleic acids toa subject in need thereof to induce immune response.

In some embodiments, the methods described herein can increase immuneresponse, activity (e.g., producing cytokines, IFN-γ, IL-2, or TNF-α),or number of immune cells (e.g., T cells, CD8+ T cells, cytotoxic Tcells, and/or CD4+ T cells) by at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, 20 folds, or50 folds. In some embodiments, the methods described herein can increaseCD107a degranulation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, 20 folds, or 50 folds.In some embodiments, the methods as described herein can increase numberof CD8+ effector T cells (e.g., total number of CD8+ effector T cells,or e.g., percentage of CD8+ in CD45+ cells) that specifically target thecancer cell or recognize the peptides described herein by at least 50%,60%, 70%, 80%, 90%, 100%, 2 folds, 3 folds, 5 folds, 10 folds, 20 folds,or 50 folds.

Nanoparticles

The disclosure further provides nanoparticles or nanocarriers comprisingone or more peptides as described herein (e.g., SEQ ID NO: 1-17) or oneor more polynucleotides as described herein (e.g., a sequence encodingSEQ ID NO: 1-17). In some cases, the nanocarriers comprise a peptidethat is identical to an amino acid sequence of SEQ ID NO: 1-17 buthaving 1 to 4 amino acid substitutions. In some instances, thesubstitutions are at one or more of positions 1, 2, and 9.Polynucleotides (e.g., mRNA, DNA) encoding such peptides can also beencapsulated in a nanocarrier. The nanoparticles or nanocarriers can beadministered to a subject in need thereof to induce immune response.

The peptides can be attached to the nanoparticles or nanocarriers viavarious attachment mechanisms. This attachment mechanism can be anelectrostatic attraction, covalent coupling, or a hydrophobicinteraction. In some embodiments, the nanoparticles can be loaded withadjuvants. The adjuvants can be a dendritic cell targeting molecule, forexample, a Toll-like receptor agonist, e.g., R-848, which is recognizedas a potent synthetic agonist of TLR7/TLR8, or an unmethylated CpGoligodeoxynucleotide, which is immunostimulatory agonist of TLR-9, ormonophosphoryl lipid A, which is immunostimulatory agonist of TLR-4, oran endosomal membrane targeting agent, e.g., the Endo-Porter peptide.

The polymer that forms the nanoparticles can be any biodegradable ornon-biodegradable synthetic or natural polymer. Preferably, the polymeris a biodegradable polymer. Examples of useful biodegradable polymersinclude polylactic acid (PLA), poly(glycolic acid) (PGA), orpoly(lactic-co-glycolic acid) (PLGA). These polymers have an establishedsafety record and can be used in human subjects (Jiang, et al., Adv.Drug Deliv. Rev., 57(3): 391-410, 2005; Aguado and Lambert,Immunobiology, 184(2-3): 113-25, 1992; Bramwell, et al., Adv. DrugDeliv. Rev., 57(9): 1247-65, 2005). Other amphiphilic poly(amino acid)nanoparticles, amphiphilic polysaccharide nanoparticles, or polyionnanoparticles can be used in the vaccine composition disclosed herein(see, Akagi et al., Adv Polym Sci. 247: 31-64, 2012). The foregoingpolymers can be used alone, as physical mixtures, or by formingcopolymers. In certain embodiments, the nanoparticles are formed by amixture of poly(lactic-co-glycolicacid)-block-poly(L-histidine)-block-poly(ethylene glycol) (PLGA-PLH-PEG)triblock copolymer; PLGA-PEG diblock copolymer, and PLA. Thesecopolymers can be synthesized using standard techniques. For example,the copolymer PLGA-PLH-PEG can be synthesized using a block end-graftingstrategy.

As used herein, a “nanoparticle” is a particle in the range of between500 nm to 0.5 nm, e.g., having a diameter that is between 50 and 500 nm,having a diameter that is between 100 and 400 nm, or having a diameterthat is between 200 and 400 nm.

Nanoparticles and how to make and use nanoparticles are known in theart, and are described, e.g., in US 2016/0008451, US 2010/0129439,US2018/0021258, each of which is incorporated herein by reference in itsentirety. In some embodiments, the nanoparticle is a liposome. In someembodiments, the nanoparticle is a polymeric particle.

The polymer that forms the nanoparticles can be any biodegradable ornon-biodegradable synthetic or natural polymer. In some embodiments, thepolymer is a biodegradable polymer. Examples of useful biodegradablepolymers include polylactic acid (PLA), poly(glycolic acid) (PGA), orpoly(lactic-co-glycolic acid) (PLGA). These polymers have an establishedsafety record and can be used in human subjects (Jiang, et al, Adv. DrugDeliv. Rev., 57(3):391-410, 2005; Aguado and Lambert, Immunobiology,184(2-3): 113-25, 1992; Bramwell, et al., Adv. Drug Deliv. Rev., 57(9):1247-65, 2005). Other amphiphilic poly(amino acid) nanoparticles,amphiphilic polysaccharide nanoparticles, or polyion nanoparticles canbe used in the composition disclosed herein (see, Akagi et al, Adv PolymSci. 247:31-64, 2012).

The polymers can be used alone, as physical mixtures, or by formingcopolymers. In some embodiments, the nanoparticles are formed by amixture of poly(lactic-co-glycolicacid)-block-poly(L-histidine)-block-poly(ethylene glycol) (PLGA-PLH-PEG)triblock copolymer; PLGA-PEG diblock copolymer, and PLA. Thesecopolymers can be synthesized using standard techniques. For example,the copolymer PLGA-PLH-PEG can be synthesized using a block end-graftingstrategy. A linear structure (e.g., PLGA-PLH-PEG) can provide thenanoparticles several characteristics compatible with extendedcirculation and charge-mediated targeting.

In some embodiments, natural polymers can be used. Examples of naturalpolymers include alginate and other polysaccharides, collagen, albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion.

Other suitable biodegradable polymers include, but are not limited to,poly(hydroxy acids), such as polymers and copolymers of lactic acid andglycolic acid, polyanhydrides, poly(ortho)esters, polyesters,polyurethanes, poly(butyric acid), poly(valeric acid),poly(caprolactone), poly(hydroxyalkanoates), andpoly(lactide-co-caprolactone).

The polymer can be a bioadhesive polymer that is hydrophilic orhydrophobic. Hydrophilic polymers include CARBOPOL™ (a high molecularweight, crosslinked, acrylic acid-based polymers manufactured byNoveon), polycarbophil, cellulose esters, and dextran.

These polymers can be obtained from sources such as Sigma Chemical Co.,St. Louis, Mo.; Polysciences, Warrenton, Pa.; Aldrich, Milwaukee, Wis.;Fluka, Ronkonkoma, N.Y.; and BioRad, Richmond, Calif., or can besynthesized from monomers obtained from these or other suppliers usingstandard techniques.

A wide variety of polymers and methods for forming polymeric matricestherefrom are known conventionally. In general, a polymeric matrixcomprises one or more polymers. Polymers can be natural or unnatural(synthetic) polymers. Polymers can be homopolymers or copolymerscomprising two or more monomers. In terms of sequence, copolymers can berandom, block, or comprise a combination of random and block sequences.Typically, polymers in accordance with the present invention are organicpolymers.

Examples of polymers suitable for use in the composition describedherein include, but are not limited to polyethylenes, polycarbonates(e.g. poly(1,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacicanhydride)), polypropylfumarates, polyamides (e.g., polycaprolactam),polyacetals, polyethers, polyesters (e.g., polylactide, polyglycolide,polylactide-co-glycolide, polycaprolactone, polyhydroxyacid (e.g.poly(-hydroxyalkanoate))), poly(orthoesters), polycyanoacrylates,polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polyureas, polystyrenes, and polyamines, polylysine,polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers.

In some embodiments, polymers in accordance with the present inventioninclude polymers that have been approved for use in humans by the U.S.Food and Drug Administration (FDA) under 21 C.F.R. § 177.2600, includingbut not limited to polyesters (e.g., polylactic acid,poly(lactic-co-glycolic acid), polycaprolactone, polyvalerolactone,poly(1,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride));polyethers (e.g., polyethylene glycol); polyurethanes;polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, polymers can be hydrophilic. For example, polymerscan comprise anionic groups (e.g., phosphate group, sulfate group,carboxylate group); cationic groups (e.g., quaternary amine group); orpolar groups (e.g., hydroxyl group, thiol group, amine group). In someembodiments, polymers can be hydrophobic. Selection of thehydrophilicity or hydrophobicity of the polymer can have an impact onthe nature of materials that are incorporated (e.g., coupled) within thesynthetic nanoparticle.

In some embodiments, polymers can be modified with one or more moietiesand/or functional groups. A variety of moieties or functional groups canbe used in accordance with the present invention. In some embodiments,polymers can be modified with polyethylene glycol (PEG), with acarbohydrate, and/or with acyclic polyacetals derived frompolysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Certainembodiments can be made using the general teachings of U.S. Pat. No.5,543,158 to Gref et al, or WO publication WO2009/051837 by Von Andrianet al.

In some embodiments, polymers can be modified with a lipid or fatty acidgroup. In some embodiments, a fatty acid group can be one or more ofbutyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group can be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, polymers can be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; PEG copolymers and copolymers oflactide and glycolide (e.g., PLA-PEG copolymers, PGA-PEG copolymers,PLGA-PEG copolymers, and derivatives thereof. In some embodiments,polyesters include, for example, poly(caprolactone),poly(caprolactone)-PEG copolymers, poly(L-lactide-co-L-lysine),poly(serine ester), poly(4-hydroxy-L-proline ester),poly[a-(4-aminobutyl)-L-glycolic acid], and derivatives thereof. Thedegradation rate of PLGA can be adjusted by altering the lacticacid:glycolic acid ratio. In some embodiments, PLGA to be used inaccordance with the present invention is characterized by a lacticacid:glycolic acid ratio of approximately 85:15, approximately 75:25,approximately 60:40, approximately 50:50, approximately 40:60,approximately 25:75, or approximately 15:85.

In some embodiments, polymers can be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidyl methacrylate copolymers,polycyanoacrylates, and combinations comprising one or more of theforegoing polymers. The acrylic polymer can comprise fully-polymerizedcopolymers of acrylic and methacrylic acid esters with a low content ofquaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g., DNA, or derivatives thereof).Amine-containing polymers such as poly(lysine) (Zauner et al., 1998,Adv. Drug Del. Rev., 30:97; and Kabanov et al, 1995, Bioconjugate Chem.,6:7), poly(ethylene imine) (PEI; Boussif et al, 1995, Proc. Natl. Acad.Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers(Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897;Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al, 1993,Bioconjugate Chem., 4:372) are positively-charged at physiological pH,form ion pairs with nucleic acids, and mediate transfection in a varietyof cell lines.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains (Putnam et al, 1999, Macromolecules, 32:3658;Barrera et al, 1993, J. Am. Chem. Soc, 115: 11010; Kwon et al, 1989,Macromolecules, 22:3250; Lim et al, 1999, J. Am. Chem. Soc, 121:5633;and Zhou et al., 1990, Macromolecules, 23:3399). Examples of thesepolyesters include poly(L-lactide-co-L-lysine) (Barrera et al, 1993, J.Am. Chem. Soc, 115: 11010), poly(serine ester) (Zhou et al, 1990,Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam et al,1999, Macromolecules, 32:3658; and Lim et al, 1999, J. Am. Chem. Soc,121:5633), and poly(4-hydroxy-L-proline ester) (Putnam et al, 1999,Macromolecules, 32:3658; and Lim et al, 1999, J. Am. Chem. Soc,121:5633).

The properties of these and other polymers and methods for preparingthem are well known in the art (see, for example, U.S. Pat. Nos.6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148;5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665;5,019,379; 5,010,167; 4,806,621; 4,638,045; and U.S. Pat. No. 4,946,929;Wang et al, 2001, J. Am. Chem. Soc, 123:9480; Lim et al, 2001, J. Am.Chem. Soc, 123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999,J. Control. Release, 62:7; and Uhrich et al, 1999, Chem. Rev., 99:3181).More generally, a variety of methods for synthesizing certain suitablepolymers are described in Concise Encyclopedia of Polymer Science andPolymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press,1980; Principles of Polymerization by Odian, John Wiley & Sons, FourthEdition, 2004; Contemporary Polymer Chemistry by Allcock et al,Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S.Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732. Each of theforgoing is incorporated herein by reference in its entirety.

In some embodiments, polymers can be linear or branched polymers. Insome embodiments, polymers can be dendrimers. In some embodiments,polymers can be substantially cross-linked to one another. In someembodiments, polymers can be substantially free of cross-links. In someembodiments, polymers can be used in accordance with the presentinvention without undergoing a cross-linking step. It is further to beunderstood that inventive synthetic nanoparticles can comprise blockcopolymers, graft copolymers, blends, mixtures, and/or adducts of any ofthe foregoing and other polymers. Those skilled in the art willrecognize that the polymers listed herein represent an exemplary, notcomprehensive, list of polymers that can be of use in accordance withthe present invention.

In some embodiments, synthetic nanoparticles can optionally comprise oneor more amphiphilic entities. In some embodiments, an amphiphilic entitycan promote the production of synthetic nanoparticles with increasedstability, improved uniformity, or increased viscosity. In someembodiments, amphiphilic entities can be associated with the interiorsurface of a lipid membrane (e.g., lipid bilayer, lipid monolayer,etc.). Many amphiphilic entities known in the art are suitable for usein making synthetic nanoparticles in accordance with the presentinvention. Such amphiphilic entities include, but are not limited to,phosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine(DPPC); dioleoylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate; diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcoholssuch as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; asurface active fatty acid, such as palmitic acid or oleic acid; fattyacids; fatty acid monoglycerides; fatty acid diglycerides; fatty acidamides; sorbitan trioleate (Span®85) glycocholate; sorbitan monolaurate(Span®20); polysorbate 20 (Tween®20); polysorbate 60 (Tween®60);polysorbate 65 (Tween®65); polysorbate 80 (Tween®80); polysorbate 85(Tween®85); polyoxyethylene monostearate; surfactin; a poloxamer; asorbitan fatty acid ester such as sorbitan trioleate; lecithin;lysolecithin; phosphatidylserine; phosphatidylinositol; sphingomyelin;phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid;cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol;stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerolricinoleate; hexadecyl stearate; isopropyl myristate; tyloxapol;poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethyleneglycol)400-monostearate; phospholipids; synthetic and/or naturaldetergents having high surfactant properties; deoxycholates;cyclodextrins; chaotropic salts; ion pairing agents; and combinationsthereof. An amphiphilic entity component can be a mixture of differentamphiphilic entities. Those skilled in the art will recognize that thisis an exemplary, not comprehensive, list of substances with surfactantactivity. Any amphiphilic entity can be used in the production ofsynthetic nanoparticles to be used in accordance with the presentinvention.

In some embodiments, synthetic nanoparticles can optionally comprise oneor more carbohydrates. Carbohydrates can be natural or synthetic. Acarbohydrate can be a derivatized natural carbohydrate. In certainembodiments, a carbohydrate comprises monosaccharide or disaccharide,including but not limited to glucose, fructose, galactose, ribose,lactose, sucrose, maltose, trehalose, cellobiose, mannose, xylose,arabinose, glucuronic acid, galactoronic acid, mannuronic acid,glucosamine, galactosamine, and neuraminic acid. In certain embodiments,a carbohydrate is a polysaccharide, including but not limited topullulan, cellulose, microcrystalline cellulose, hydroxypropylmethylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC),dextran, cyclodextrin, glycogen, hydroxyethylstarch, carrageenan,glycon, amylose, chitosan, N,O-carboxymethylchitosan, algin and alginicacid, starch, chitin, inulin, konjac, glucomannan, pustulan, heparin,hyaluronic acid, curdlan, and xanthan. In embodiments, the inventivesynthetic nanoparticles do not comprise (or specifically exclude)carbohydrates, such as a polysaccharide. In certain embodiments, thecarbohydrate can comprise a carbohydrate derivative such as a sugaralcohol, including but not limited to mannitol, sorbitol, xylitol,erythritol, maltitol, and lactitol.

In some embodiments, the nanoparticle comprises a peptide comprising asequence set forth in SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 16(e.g., SEQ ID NO: 13). In certain instances, the nanoparticles disclosedherein can be administered in combination with another therapy describedbelow (e.g., APC-based therapy, CTL-based therapy, peptide vaccinetherapy). In some instances, the nanoparticles disclosed herein can beadministered to a human subject in combination with an immune agonist(e.g., anti-OX40 antibody; anti-GITR antibody), a checkpoint inhibitor(e.g., anti-LAG3 antibody), and/or lenalidomide.

Antigen Presenting Cell (APC)-Based Immunotherapy

An ex vivo strategy for inducing an immune response in a subject caninvolve contacting suitable antigen presenting cells (e.g., dendriticcells, monocytes, or macrophages) obtained from the subject with any ofthe peptides described herein. Alternatively, the cells can betransfected with a nucleic acid (e.g., an expression vector) encodingone or more of the peptides and optionally cultured for a period of timeand under conditions that permit the expression of the peptides. Thetransfection method will depend on the type of cell and nucleic acidbeing transfected into the cell. Following the contacting ortransfection, the cells are then returned to the subject.

The cells can be any of a wide range of types expressing MHC class I orII molecules. For example, the cells can include bone marrow cells,macrophages, monocytes, dendritic cells, T cells (e.g., T helper cells,CD4+ cells, CD8+ cells, or cytotoxic T cells), or B cells.

Thus, the disclosure provides a composition comprising an APC (e.g.,dendritic cell), wherein the APC presents a peptide sequence on itssurface, wherein the peptide sequence comprises at least one majorhistocompatibility complex (WIC) class I or class II peptide epitope ofone or both of BCMA antigen (SEQ ID NO: 18) and TACI antigen (SEQ ID NO:19). In some embodiments, the APC is a dendritic cell. In someembodiments, the MHC peptide epitope is MHC class I peptide epitope(e.g., HLA-A2 or HLA-A24 peptide epitope). In some embodiments, the APCacquires the peptide sequence in vitro by exposure to a syntheticpeptide comprising the peptide sequence.

In some embodiments of any of the ex vivo methods, cells that areobtained from the subject, or from a subject of the same species otherthan the subject (allogeneic) can be contacted with the reagents (orimmunogenic/antigenic compositions) and administered to the subject.

In some embodiments, the composition comprises at least 10⁴, 10⁵, 10⁶,10⁷, 10⁸, or 10⁹ APCs (e.g., dendritic cells). In some embodiments, thecomposition comprises less than 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ APCs(e.g., dendritic cells).

a. Preparation of Antigen Presenting Cells

Antigen presenting cells (APC), such as dendritic cells (DC), suitablefor administration to subjects (e.g., multiple myeloma patients) can beisolated or obtained from any tissue in which such cells are found, orcan be otherwise cultured and provided. APC (e.g., DC) can be found, byway of example, in the bone marrow or peripheral blood mononuclear cells(PBMC) of a mammal, in the spleen of a mammal or in the skin of a mammal(i.e., Langerhan's cells, which possess certain qualities similar tothat of DC, can be found in the skin). For instance, bone marrow can beharvested from a mammal and cultured in a medium that promotes thegrowth of DC. GM-CSF, IL-4 and/or other cytokines (e.g., TNF-α), growthfactors and supplements can be included in this medium. After a suitableamount of time in culture in medium containing appropriate cytokines(e.g., suitable to expand and differentiate the DCs into mature DCs,e.g., 4, 6, 8, 10, 12, or 14 days), clusters of DC cultured in thepresence of antigens of interest (e.g., in the presence of one or morepeptide epitopes of BCMA or TACI, or a combination of at least twopeptides of SEQ ID NOs: 13-17) and harvested for use in a cancer vaccineusing standard techniques. Antigens (e.g., isolated or purifiedpeptides, or synthetic peptides) can be added to cultures at aconcentration of 1 μg/ml-50 μg/ml per antigen, e.g., 2, 5, 10, 20, 30,or 40 μg/ml per antigen.

In some embodiments, APC are isolated from a subject (e.g., a human).Mononuclear cells are isolated from blood using leukapheresis (e.g.,using a COBE Spectra Apheresis System). The mononuclear cells areallowed to become adherent by incubation in tissue culture flasks for 2hours at 37° C. Non-adherent cells are removed by washing. Adherentcells are cultured in medium supplemented with granulocyte macrophagecolony stimulating factor (GM-CSF) (800 units/ml, clinical grade,Immunex, Seattle, Wash.) and interleukin-4 (IL-4)(500 units/ml, R&DSystems, Minneapolis, Minn.) for five days. On day five, TNF-α is addedto the culture medium for another 3-4 days. On day 8 or 9, cells areharvested and washed, and incubated with peptide antigens for 16-20hours on a tissue rotator. Peptide antigens are added to the cultures ata concentration of ^(˜)10 μg/ml (per antigen).

Various other methods can be used to isolate the APCs, as would berecognized by one of skill in the art. DCs occur in low numbers in alltissues in which they reside, making isolation and enrichment of DCs arequirement. Any of a number of procedures entailing repetitive densitygradient separation, fluorescence activated cell sorting techniques,positive selection, negative selection, or a combination thereof areroutinely used to obtain enriched populations of isolated DCs. Guidanceon such methods for isolating DCs can be found in O'Doherty, U. et al.,J. Exp. Med., 178: 1067-1078, 1993; Young and Steinman, J. Exp. Med.,171: 1315-1332, 1990; Freudenthal and Steinman, Proc. Nat. Acad. Sci.USA, 57: 7698-7702, 1990; Macatonia et al., Immunol., 67: 285-289, 1989;Markowicz and Engleman, J. Clin. Invest., 85: 955-961, 1990;Mehta-Damani et al., J. Immunol., 153: 996-1003, 1994; and Thomas etal., J. Immunol., 151: 6840-6852, 1993. One method for isolating DCsfrom human peripheral blood is described in U.S. Pat. No. 5,643,786.

The dendritic cells prepared according to methods described hereinpresent epitopes corresponding to the antigens at a higher averagedensity than epitopes present on dendritic cells exposed to a tumorlysate (e.g., a neural tumor lysate). The relative density of one ormore antigens on antigen presenting cells can be determined by bothindirect and direct means. Primary immune response of naïve animals isroughly proportional to antigen density of antigen presenting cells(Bullock et al., J. Immunol., 170: 1822-1829, 2003). Relative antigendensity between two populations of antigen presenting cells cantherefore be estimated by immunizing an animal with each population,isolating B or T cells, and monitoring the specific immune responseagainst the specific antigen by, e.g., tetramer assays, ELISPOT, orquantitative PCR.

Relative antigen density can also be measured directly. In one method,the antigen presenting cells are stained with an antibody that bindsspecifically to the WIC-antigen complex, and the cells are then analyzedto determine the relative amount of antibody binding to each cell (see,e.g., Gonzalez et al., Proc. Natl. Acad. Sci. USA, 102: 4824-4829,2005). Exemplary methods to analyze antibody binding include flowcytometry and fluorescence activated cell sorting. The results of theanalysis can be reported e.g., as the proportion of cells that arepositive for staining for an individual MHC-antigen complex or theaverage relative amount of staining per cell. In some embodiments, ahistogram of relative amount of staining per cell can be created.

In some embodiments, antigen density can be measured directly by directanalysis of the peptides bound to WIC, e.g., by mass spectrometry (see,e.g., Purcell and Gorman, Mol. Cell. Proteomics, 3: 193-208, 2004).Typically, WIC-bound peptides are isolated by one of several methods. Inone method, cell lysates of antigen presenting cells are analyzed, oftenfollowing ultrafiltration to enrich for small peptides (see, e.g., Falket al., J. Exp. Med., 174: 425-434, 1991; Rotzxhke et al., Nature, 348:252-254, 1990). In another method, WIC-bound peptides are isolateddirectly from the cell surface, e.g., by acid elution (see, e.g.,Storkus et al., J. Immunother., 14: 94-103, 1993; Storkus et al., J.Immunol., 151: 3719-27, 1993). In another method, MHC-peptide complexesare immunoaffinity purified from antigen presenting cell lysates, andthe MHC-bound peptides are then eluted by acid treatment (see, e.g.,Falk et al., Nature, 351: 290-296). Following isolation of MI-IC-boundpeptides, the peptides are then analyzed by mass spectrometry, oftenfollowing a separation step (e.g., liquid chromatography, capillary gelelectrophoresis, or two-dimensional gel electrophoresis). The individualpeptide antigens can be both identified and quantified using massspectrometry to determine the relative average proportion of eachantigen in a population of antigen presenting cells. In some methods,the relative amounts of a peptide in two populations of antigenpresenting cells are compared using stable isotope labeling of onepopulation, followed by mass spectrometry (see Lemmel et al., Nat.Biotechnol., 22: 450-454, 2004).

b. Administration of Antigen Presenting Cells

The APC-based cancer vaccine may be delivered to a patient or testanimal by any suitable delivery route, which can include injection,infusion, inoculation, direct surgical delivery, or any combinationthereof. In some embodiments, the cancer vaccine is administered to ahuman in the deltoid region or axillary region. For example, the vaccineis administered into the axillary region as an intradermal injection. Insome embodiments, the vaccine is administered intravenously.

An appropriate carrier for administering the cells may be selected byone of skill in the art by routine techniques. For example, thepharmaceutical carrier can be a buffered saline solution, e.g., cellculture media, and can include DMSO for preserving cell viability.

The quantity of APC appropriate for administration to a patient as acancer vaccine to effect the methods of the present invention and themost convenient route of such administration may be based upon a varietyof factors, as may the formulation of the vaccine itself. Some of thesefactors include the physical characteristics of the patient (e.g., age,weight, and sex), the physical characteristics of the tumor (e.g.,location, size, rate of growth, and accessibility), and the extent towhich other therapeutic methodologies (e.g., chemotherapy, and beamradiation therapy) are being implemented in connection with an overalltreatment regimen. Notwithstanding the variety of factors one shouldconsider in implementing the methods of the present invention to treat adisease condition, a mammal can be administered with from about 10⁵ toabout 10⁸ APC (e.g., 10⁷ APC) in from about 0.05 mL to about 2 mLsolution (e.g., saline) in a single administration. Additionaladministrations can be carried out, depending upon the above-describedand other factors, such as the severity of tumor pathology. In oneembodiment, from about one to about five administrations of about 10⁶APC is performed at two-week intervals.

DC vaccination can be accompanied by other treatments. For example, apatient receiving DC vaccination may also be receiving chemotherapy,radiation, and/or surgical therapy concurrently. Methods of treatingcancer using DC vaccination in conjunction with chemotherapy aredescribed in Wheeler et al., US Pat. Pub. No. 2007/0020297. In someembodiments, a patient receiving DC vaccination has already receivedchemotherapy, radiation, and/or surgical treatment for the cancer. Inone embodiment, a patient receiving DC vaccination is treated with aCOX-2 inhibitor, as described in Yu and Akasaki, WO 2005/037995.

T Cell-Based Immunotherapy

Ex vivo methods for stimulating an immune response can also includecontacting in vitro a T cell (e.g., in a population of lymphocytesobtained from a subject) with an antigen-presenting cell (APC)expressing an MHC molecule bound to one of the peptides described hereinfor an amount of time (and under conditions) that is sufficient toactivate the T cell (e.g., cytotoxic T cells and/or CD4+ helper Tcells). Thus, the disclosure provides methods of generating and/orproliferating BCMA-specific and/or TACI-specific T cells (e.g.,cytotoxic T cells and/or CD4+ helper T cells). The methods involvecontacting one or more T cells (e.g., cytotoxic T cells and/or CD4+helper T cells) with one or more antigen presenting cells pulsed with apeptide as described herein. These T cells can be cytotoxic T cells,e.g., memory cytotoxic T cells, effector cytotoxic T cells, or CD4+helper T cells.

The activated T cells can be used kill a target cell. In someembodiments, the methods involve contacting the target cell with one ormore BCMA-specific cytotoxic T cells, wherein the target cell expressesor overexpresses BCMA, and expresses HLA-A. In some embodiments, themethods involve contacting the target cell with one or moreTACI-specific cytotoxic T cells, wherein the target cell expresses oroverexpresses TACI, and expresses HLA-A.

In some embodiments, the BCMA- or TACI-specific T cells (e.g., cytotoxicT cells and/or CD4+ helper T cells) are administered in combination witha peptide disclosed herein (e.g., one or more of SEQ ID NOs: 13-17), anAPC that presents a BCMA (e.g., SEQ ID NO: 13 or 14) or TACI (SEQ ID NO:16) peptide, lenalidomide, an immunomodulatory agent, a checkpointinhibitor (e.g., anti-LAG3 antibody) or an immune agonist (e.g.,anti-OX40, anti-GITR). In some embodiments, the additional therapeuticagent administered with the BCMA- or TACI-specific CTL T cells is anantibody (e.g., human antibody) the specifically binds to PD-1, CTLA-4,LAG-3, BTLA, PD-L1, CD27, CD28, CD40, CD47, 4-1BB (CD137), CD154, TIGIT,TIM-3, GITR (CD357), OX40, CD20, EGFR, or CD319. In some embodiments,the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-L1antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGITantibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or ananti-GITR antibody. In some embodiments, the T cells are administered incombination with an immune agonist, e.g., an anti-OX40 or anti-GITRantibody.

The activated T cell(s) can also be reintroduced into the subject fromwhich the cells were obtained. In some embodiments, T cells can beobtained from a subject of the same species other than the subject(allogeneic) can be contacted with the reagents (orimmunogenic/antigenic compositions) and administered to the subject.

In some embodiments, T cells are derived from in vitro induction inpatient-derived peripheral blood mononuclear cells (PBMC). The followingprotocol can be used to produce antigen specific CTL in vitro frompatient derived PBMC. To generate dendritic cells, the plastic adherentcells from PBMCs are cultured in AIM-V medium supplemented withrecombinant human GM-CSF and recombinant human IL-4 at 37° C. in ahumidified CO₂ (5%) incubator. Six days later, the immature dendriticcells in the cultures are stimulated with recombinant human TNF-α formaturation. Mature dendritic cells are then harvested on day 8,resuspended in PBS at 1×10⁶ per mL with peptide (2 μg/mL), and incubatedfor 2 hours at 37° C. Autologous CD8+ T cells are enriched from PBMCsusing magnetic microbeads (Miltenyi Biotec, Auburn, Calif.). CD8+ Tcells (2×10⁶ per well) are cocultured with 2×10⁵ per well peptide-pulseddendritic cells in 2 mL/well of AIM-V medium supplemented with 5% humanAB serum and 10 units/mL rhIL-7 (Cell Sciences) in each well of 24-welltissue culture plates. About 20 U/ml of IL-2 is added 24 h later atregular intervals, 2 days after each restimulation. On day 7,lymphocytes are restimulated with autologous dendritic cells pulsed withpeptide in AIM-V medium supplemented with 5% human AB serum, rhIL-2, andrhIL-7 (10 units/mL each). About 20 U/ml of IL-2 is added 24 h later atregular intervals, 2 days after each restimulation. On the seventh day,after the three rounds of restimulation, cells are harvested and testedthe activity of CTL. The stimulated CD8+ cultured cells (CTL) areco-cultured with T2 cells (a human TAP-deficient cell line) pulsed with2 μg/ml Her-2, gp100, AIM-2, MAGE-1, or IL13 receptor α2 peptides. After24 hours incubation, IFN-γ in the medium is measured by ELISA assay.

Chimeric Antigen Receptor (CAR) T-Cell Based Immunotherapy

The present disclosure further provides methods for adoptive transfer ofT cells expressing chimeric antigen receptors for treating a cancer.CAR-modified T cells can be engineered to target virtually any tumorassociated antigen (e.g., BCMA and/or TACI). Usually, T cells aregenetically engineered to express CARs specifically directed towardsantigens on the patient's tumor cells, then infused back into thepatient.

The common form of CARs are fusions of single-chain variable fragments(scFv), fused to CD3-zeta transmembrane- and endodomain. The scFV can bederived from the antigen-specific receptor of T cells (e.g.,BCMA-specific cytotoxic T cells, or TACI-specific cytotoxic T cells), orantibodies that specifically bind to the antigen.

In some embodiments, the sequence of the T cell receptors inBCMA-specific cytotoxic T cells or TACI-specific cytotoxic T cells isdetermined, e.g., by sequencing. The sequence of the T cell receptors inBCMA-specific cytotoxic T cells and/or TACT-specific cytotoxic T cellscan be used to generate a CAR.

In some embodiments, these T cells are collected from the patient. Insome embodiments, these T cells are obtained from induced pluripotentstem cell (iPSC).

Viral vectors such as retrovirus, lentivirus or transposon, are oftenused to integrate the transgene (e.g., CAR) into the host cell genome.Alternatively, non-integrating vectors such as plasmids or mRNA can beused to transfer the CAR gene to the T cells, and make T cells toexpress CAR under appropriate conditions.

Induced Pluripotent Stem Cell-Approaches

Adoptive T-cell therapy with the administration of a large number of exvivo expanded activated antigen-specific cytotoxic T lymphocytes (CTL)targeting tumor specific-antigens has induced durable remissions inselected malignancies. Although utilizing TCR which recognize mainlyintracellular antigens that have already been processed and presented aspeptide complexes with MHC molecules (Johnson et al. 2009; Morgan et al.2006) may further enhance tumor selectivity, introduction of exogenousTCR genes can result in mismatching of transferred and endogenous α andβ chains, resulting in serious autoimmune adverse events (Bendle et al.2010, Hinrichs et al. 2013). In contrast, CAR-T recognize antigensexpressed on the cell surface in a non-MHC-restricted manner. To date,the most successful CAR-T therapy targeting the B-cell antigen CD19 hasachieved minimal residual disease negative complete responses inpatients with relapsed and chemo-refractory B-cell malignancies(Kochenderfer et al. 2010, Grupp et al. 2013). Nonetheless ongoingefforts are directed to minimize adverse effects, including cytokinerelease syndrome, and improve durability of response (Brentjens et al.2011, Kalos et al. 2011, Kochenderfer et al. 2012, Porter et al. 2011).Importantly, CTL continuously exposed to tumor antigens during long-termexpansion to be used for TCR-based or CAR-based therapy, may lose theirproliferative capacity (“exhausted”) and their functional activity withterminal differentiation.

To overcome these limitations, a technique currently being developed isexploitation of fully rejuvenated CTL from “induced pluripotent stemcells (iPSC)”. These iPSC are a special type of pluripotent cell thatare derived from adult somatic cells upon ectopic expression of a set ofdefined transcription factors. Importantly, tumor antigen-specific CTLcan be reprogrammed by iPSC technology from antigen-specific CTL(Vizcardo et al. 2013, Ando et al. 2015, Timmermans et al. 2009, Kennedyet al. 2012). These iPSC-CTL are functionally rejuvenated anddemonstrate longer telomeres (1.5 fold increase) and a higherproliferative capacity (5-50 fold increase) than the original CTL fromwhich they were derived (Nishimura et al. 2013). This powerfulreprogramming therapeutic approach has the potential to markedlyincrease the efficacy and durability of antigen-specific cancerimmunotherapy. Thus, the disclosure provides methods of rejuvenatingcytotoxic T cells. In some embodiments, the methods can increase theproliferative capacity by at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50,60, 70, 80, 90, or 100 folds.

Activation of tumor-specific CTLs is the main goal of many cancerimmunotherapies. The isolation of tumor-specific T-cells from a cancerpatient, in vitro preparation (activation and expansion), andtransfusion of these T-cells to the patient are basic steps of adaptiveimmunotherapy with T-cell. iPSC technology can be used to improve theefficacy of adoptive cell transfer immunotherapy (ACT).

The iPSC can be obtained from differentiated cells (e.g., fibroblasts,immune cells, T cells, B cells) induced through retroviral transfectionof Yamanaka factors (a combination of Oct3/4, Sox2, Klf4, and c-Myc),and differentiated into T-cell lineages by culturing it on monolayerOP9-DL1 cell system in addition to Flt-3 ligand and IL-7.

In some embodiments, iPSCs can be generated from T-cells. After theexpansion, these cells are differentiated again into T-cells. Human Tlymphocyte can act as cell source for iPSC generation. Peripheral bloodmononuclear cells (PBMCs) can be separated from whole blood byleukapheresis or venipuncture and then CD3+ T-cells can be expanded bystimulation with IL-2 and anti-CD3 antibody. T-cell-derived iPSCs (TiPS)can be generated from activated T-cell when exposed to retroviraltransduction of the reprogramming factors. These T-iPSCs preserve theiroriginal T-cell receptor (TCR) gene rearrangements, so they can be usedas an unlimited source of hematopoietic stem cells bearing endogenoustumor-specific TCR gene for cancer ACT therapy.

Thus, in some embodiments, iPSCs are generated from antigen-specificcytotoxic T cells. These antigen-specific T cells are generated by themethods as described herein, e.g., by contacting one or more T cellswith one or more antigen presenting cells pulsed with a peptidecomprising an amino acid sequence as described herein (e.g., SEQ ID NOs:1-17). As the T-iPSCs preserve their original T-cell receptor (TCR) generearrangements, after these T-iPSCs differentiates into T cells, these Tcells can recognize BCMA and/or TACI on a cancer cell.

In some embodiments, a nucleic acid that encodes CAR that specificallyrecognizes BCMA and/or TACI can be introduced into T-iPSCs. Once afterthese T-iPSCs differentiates into T cells, these T cells can recognizeBCMA and/or TACI on a cancer cell.

In some embodiments, the differentiated T cells are administered to asubject. In some embodiments, T-iPSCs are administered to a subject, andthen these cells are differentiated into cytotoxic T cells in the bodyof the subject.

Subjects

The subject can be any animal capable of an immune response to anantigen. The terms “subject” and “patient” are used interchangeablythroughout the specification and describe an animal, human or non-human,to whom treatment according to the methods of the present disclosure isprovided. Veterinary and non-veterinary applications are contemplated bythe present invention. Human patients can be adult humans or juvenilehumans (e.g., humans below the age of 18 years old). In addition tohumans, subjects include but are not limited to mice, rats, hamsters,guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are,for example, non-human primates (e.g., monkey, chimpanzee, gorilla, andthe like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets,rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine, canine,feline, bovine, and other domestic, farm, and zoo animals.

The subject can be one having, suspected of having, or at risk ofdeveloping a cancer. As used herein, the term “cancer” refers to cellshaving the capacity for autonomous growth, i.e., an abnormal state orcondition characterized by rapidly proliferating cell growth. The termis meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. The term “tumor” as used herein refers to cancerous cells,e.g., a mass of cancerous cells. Cancers that can be treated ordiagnosed using the methods described herein include malignancies of thevarious organ systems, such as affecting lung, breast, thyroid,lymphoid, gastrointestinal, and genito-urinary tract, as well asadenocarcinomas which include malignancies such as most colon cancers,renal-cell carcinoma, prostate cancer and/or testicular tumors,non-small cell carcinoma of the lung, cancer of the small intestine andcancer of the esophagus. In some embodiments, the agents describedherein are designed for treating or diagnosing a carcinoma in a subject.The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. In some embodiments, thecancer is renal carcinoma or melanoma. Exemplary carcinomas includethose forming from tissue of the cervix, lung, prostate, breast, headand neck, colon and ovary. The term also includes carcinosarcomas, e.g.,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures. The term “sarcoma” is art recognized and refers to malignanttumors of mesenchymal derivation. In some embodiments, the subject has ahematological cancer, e.g., multiple myeloma, leukemia, non-Hodgkinlymphoma, or Hodgkin lymphoma.

In some embodiments, the subject has a BCMA-expressing/overexpressingdisease or a TACI-expressing/overexpressing disease, including e.g.,multiple myeloma, B cell-related malignancies, plasma cell-relatedmalignancies, a pre-malignant disease (e.g., a pre-malignant disease ofMM, such as SMM or MGUS).

In some embodiments, the subject can be one having, suspected of having,or at risk of developing a plasma cell disorder. As used herein, theterm “plasma cell disorders” refer to a group of diseases or disorderscharacterized by clonal plasma cell (PC) proliferation andhyper-secretion of paraproteins (e.g., monoclonal immunoglobulin and/orfree light chain (FLC)).

Non-limiting examples of plasma cell disorders include monoclonalgammopathy of undermined significance (MGUS), multiple myeloma (MM),Waldenström macroglobulinemia (WM), light chain amyloidosis (AL),solitary plasmacytoma (e.g., solitary plasmacytoma of bone, orextramedullary plasmacytoma), polyneuropathy, organomegaly,endocrinopathy monoclonal gammopathy and skin changes syndrome (POEMS),and heavy-chain disease. Other plasm cell disorders include, e.g.,Monoclonal Gammopathy of Renal Significance (MGRS), MGUS-associatedneuropathy, and other paraproteinemic neuropathy.

MGUS, smoldering MM (SMM), and symptomatic MM represent a spectrum ofthe same disease. Symptomatic or active multiple myeloma ischaracterized by more than 10% BM infiltration by clonal plasma cellsand/or biopsy proven plasmacytoma in addition to any level of monoclonalprotein and the presence of end-organ damage that consists of a myelomadefining event in the form of any of the CRAB criteria (hypercalcemia,renal insufficiency, anemia, or bone lesions which are deemed related tothe plasma cell clone) or any of the new biomarker of malignancy (BMinvolvement by equal or greater than 60% clonal plasma cell; a ratio ofinvolved versus uninvolved FLC equal or exceeding 100; and/or thepresence of more than one bone lesion on MRI (Kyle R. A. et al.,Leukemia, 23: 3-9 (2009); Rajkumar V. S. et al, Lancet Oncology, 15: 12,2014). MM is a plasma cell malignancy that characteristically involvesextensive infiltration of bone marrow (BM), and occasionally theformation of plasmacytoma, as discrete clusters of malignant plasmacells inside or outside of the BM space (Kyle R. A. et al., N. Engl. J.Med., 351: 1860-73 (2004)). Consequences of this disease are numerousand involve multiple organ systems. Disruption of BM and normal plasmacell function leads to anemia, leukopenia, hypogammaglobulinemia, andthrombocytopenia, which variously result in fatigue, increasedsusceptibility to infection, and, less commonly, increased tendency tobleed. Disease involvement in bone creates osteolytic lesions, producesbone pain, and may be associated with hypercalcemia (Kyle R. A. et al.,Blood, 111: 2962-72 (2008)).

Smoldering MM (SMM) is characterized by having a serum immunoglobulin(Ig) G or IgA monoclonal protein of 30 g/L or higher and/or 10% or moreplasma cells in the bone marrow but no evidence of end-organ damage ormalignancy-defining biomarkers (Rajkumar et al, Lancet, 2014). A studyof the natural history of SMM suggests that there are 2 different types:evolving smoldering MM and non-evolving Smoldering MM (Dimopoulos M. etal., Leukemia, 23(9): 1545-56 (2009)). Evolving SMM is characterized bya progressive increase in M protein and a shorter median time toprogression (TTP) to active multiple myeloma of 1.3 years. Non-evolvingSMM has a more stable M protein that may then change abruptly at thetime of progression to active multiple myeloma, with a median TTP of 3.9years.

Monoclonal gammopathy of undetermined significance (MGUS), is acondition in which an abnormal immunoglobin protein (known as aparaprotein) is found in the blood during standard laboratory bloodtests. MGUS resembles multiple myeloma and similar diseases, but thelevels of antibody are lower, the number of plasma cells (white bloodcells that secrete antibodies) in the bone marrow is lower, and it hasno symptoms or major problems.

In some embodiments, the subject has multiple myeloma, SMM, or MGUS. Insome embodiments, the subject can be one in remission from multiplemyeloma. In some embodiments, the subject has a pre-malignant disease(e.g., a pre-malignant disease of MM, such as SMM or MGUS).

In some embodiments, the subject can have a type of cancer thatexpresses or overexpress BCMA or TACI. Thus, the methods can alsoinclude the step of, prior to administering the one or more peptides (ornucleic acids) to the subject, determining whether one or more cancercells of the subject's cancer (e.g., multiple myeloma) express oroverexpress BCMA or TACI. Expression of these proteins includes bothmRNA and protein expression. Methods for detecting protein and mRNAexpression in a cell are known in the art and include, e.g.,enzyme-linked immunosorbent assay (ELISA), western and dot-blottingtechniques, or immunohistochemistry techniques for detecting protein andreverse transcription-polymerase chain reaction (RT-PCR) ornorthern-blotting techniques for detecting mRNA. In some embodiments,the average level of expression of BCMA or TACI in the cancer cell is atleast 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% higher than theaverage level of expression of BCMA or TACI in a normal cell (e.g., anormal tissue cell in the same subject, a normal plasma cell in the samesubject, or a tissue cell or a plasma cell in a healthy subject). Insome embodiments, the average level of expression of BCMA or TACI in thecancer cell is at least 2 fold, 3 fold, 5 fold, 10 fold, 20 fold, or 50fold higher than the average level of expression of BCMA or TACI in anormal cell (e.g., a normal tissue cell in the same subject, a normalplasma cell in the same subject, or a tissue cell or a plasma cell in ahealthy subject).

The subject can have, be suspected of having, or be at risk ofdeveloping a cancer (e.g., multiple myeloma). A subject “suspected ofhaving a cancer” is one having one or more symptoms of a cancer.Symptoms of cancer are well-known to those of skill in the art andgenerally include, without limitation, pain, weight loss, weakness,excessive fatigue, difficulty eating, loss of appetite, chronic cough,worsening breathlessness, coughing up blood, blood in the urine, bloodin stool, nausea, vomiting, abdominal fullness, bloating, fluid inperitoneal cavity, vaginal bleeding, constipation, abdominal distension,perforation of colon, acute peritonitis (infection, fever, pain), pain,vomiting blood, heavy sweating, fever, high blood pressure, anemia,diarrhea, jaundice, dizziness, chills, muscle spasms, difficultyswallowing, and the like. Symptoms of multiple myeloma specificallyinclude, e.g., bone pain (e.g., in the back or ribs), high levels ofcalcium in the blood, excessive thirst or urination, constipation,nausea, loss of appetite, confusion, weakness or numbness in the legs,weight loss, or repeated infections.

As used herein, a subject “at risk of developing a cancer” is a subjectthat has a predisposition to develop a cancer, i.e., a geneticpredisposition to develop cancer such as a mutation in a tumorsuppressor gene (e.g., mutation in BRCA1, p53, RB, or APC), has beenexposed to conditions, or is presently affected by conditions, that canresult in cancer. Thus, a subject can also be one “at risk of developinga cancer” when the subject has been exposed to mutagenic or carcinogeniclevels of certain compounds (e.g., carcinogenic compounds in cigarettesmoke such as acrolein, 4-aminobiphenyl, aromatic amines, benzene, beno{a}anthracene, benzo{a}pyrene, formaldehyde, hydrazine, Polonium-210(Radon), urethane, or vinyl chloride). The subject can be “at risk ofdeveloping a cancer” when the subject has been exposed to, e.g., largedoses of ultraviolet light or X-irradiation, or exposed (e.g., infected)to a tumor-causing/associated virus such as papillomavirus, Epstein-Barrvirus, hepatitis B virus, or human T-cell leukemia-lymphoma virus. Inaddition, a subject can be “at risk of developing a cancer” when thesubject suffers from an inflammation (e.g., chronic inflammation). Asubject can be at risk of developing multiple myeloma if, e.g., thesubject has monoclonal gammopathy of undetermined significance (MGUS).Thus, it is understood that subjects “suspected of having a cancer” or“at risk of developing a cancer” are not all the subjects within aspecies of interest.

In some embodiments, the methods can also include the step ofdetermining whether a subject has a cancer. Suitable methods for such adetermination depend on the type of cancer to be detected in thesubject, but are known in the art. Such methods can be qualitative orquantitative. For example, a medical practitioner can diagnose a subjectas having multiple myeloma when the subject exhibits two or more (e.g.,three, four, five, or six or more) symptoms of multiple myeloma such asany of those described herein. A subject can also be determined to havemultiple myeloma by measuring the blood calcium level, the white or redblood cell count, or the amount of protein in the urine of a subject.

MHC Molecule Multimer

The disclosure also features compositions comprising: (i) one or more ofany of the peptides described herein and (ii) a major histocompatibilitycomplex (MHC) molecule multimer. The multimer contains two or more(e.g., three, four, five, six, seven, eight, nine, ten or more) entireMHC molecules or peptide-binding regions of an WIC molecule. The one ormore peptides can be associated with (e.g., covalently or non-covalentlybound to) the WIC molecule multimer.

An MHC molecule of the multimer can be an WIC class I molecule (e.g., anHLA-A2 molecule) or an MHC class II molecule. The MHC molecule can be amammalian (e.g., a rodent, a non-human primate, a human, or any othermammal described herein) MHC molecule.

The two or more MHC molecules (or the peptide-binding regions of the WICmolecules) in the multimer can be from the same WIC molecule or fromdifferent WIC molecules. For example, an MHC molecule multimer cancontain five WIC molecules, three of which are the same MHC moleculesand two of which are different from the first three. In another example,each MHC molecule of the multimer is different. At least one of the MHCmolecules can bind to at least one of the peptides.

In some embodiments, the above compositions can contain at least two(e.g., two, three, four, five, six, seven, eight, nine, 10, 11, or 15 ormore) of any of the peptides described herein.

The compositions can also be associated with a detectable label. Forexample, one or more of the MHC molecules of the multimer can becovalently or non-covalently bound to a detectable label. Suitabledetectable labels (e.g., enzymes, fluorescent materials, luminescentmaterials, bioluminescent materials, or radionuclides) as well asmethods for joining detectable labels to a peptide or an MHC moleculeare also provided.

An MHC multimer composition can be generated using a peptide describedherein as follows: a peptide that binds to an HLA molecule is refoldedin the presence of the corresponding HLA heavy chain andβ2-microglobulin to generate a trimolecular complex. The complex is thenbiotinylated at the carboxyl terminal end of the heavy chain at a sitethat was previously engineered into the heavy chain. Multimer formationis then induced by the addition of streptavidin.

As T cell receptors are capable of recognizing a specific peptide-WICcomplex on a target cell among a wide variety of other peptide-MHCcomplexes, the MHC multimer compositions described herein can be usedto, e.g., detect antigen-specific T cells in a population of unrelated Tcells. For such assays, the multimers will generally be detectablylabeled.

For example, a multimeric MHC molecule/peptide complex can be used in anassay to assess peripheral blood mononuclear cells for the presence ofantigen-specific CTL following exposure to an immunogen. The WICmultimer complex can be used to directly visualize antigen-specific CTL(see, e.g., Ogg et al., Science 279: 2103-2106, 1998; and Altman et al.,Science 174: 94-96, 1996) and determine the frequency of theantigen-specific CTL population in a sample of peripheral bloodmononuclear cells. In some embodiments, a detectably-labeledstreptavidin used to multimerize the WIC multimer can be used to label Tcells that bind to the MHC molecule/peptide complexes of the multimer.To do this, cells treated with the multimer are exposed, e.g., to alabel (e.g., a fluorophore conjugated to biotin). The cells can then bereadily isolated or detected, e.g., using flow cytometry.

The peptides (and pharmaceutical compositions thereof), MHC multimercontaining compositions, kits, and articles of manufacture describedherein can be used in a variety of methods. For example, the peptidescan be used to: (i) induce an immune response in a subject (e.g., asubject with a cancer); (ii) activate a T cell in culture; and/or (iii)treat or event prevent a cancer such as multiple myeloma. As describedherein, the MHC multimer containing compositions can be used to, e.g.,detect antigen-specific T cells in a population of unrelated T cells.

Methods for Producing an Antibody in a Subject

Methods of producing an antibody specific for an immunogen (e.g., one ormore of any of the peptides described herein) are known in the art. Forexample, antibodies or antibody fragments specific for a peptidedescribed herein can be generated by immunization, e.g., using ananimal, or by in vitro methods such as phage display. All or part of apeptide described herein can be used to generate an antibody or antibodyfragment.

A peptide can be used to prepare antibodies by immunizing a suitablesubject, (e.g., rabbit, goat, mouse, or other mammal such as a human)with the peptide. An appropriate immunogenic preparation can contain,for example, any of the composition described herein. The preparationcan further include an adjuvant, such as Freund's complete or incompleteadjuvant, alum, RIBI, or similar immunostimulatory agent. Adjuvants alsoinclude, e.g., cholera toxin (CT), E. coli heat labile toxin (LT),mutant CT (MCT) (Yamamoto et al. (1997) J. Exp. Med. 185: 1203-1210) andmutant E. coli heat labile toxin (MLT) (Di Tommaso et al. (1996) Infect.Immunity 64: 974-979). MCT and MLT contain point mutations thatsubstantially diminish toxicity without substantially compromisingadjuvant activity relative to that of the parent molecules. Immunizationof a suitable subject with an immunogenic peptide preparation (e.g., anyof the compositions described herein) induces a polyclonal anti-peptideantibody response. In some embodiments, a toll like receptor-3 ligand(e.g., Poly ICLC), interferon alfa (IFNα), interferon gamma (IFNγ), orGranulocyte-macrophage colony-stimulating factor (GM-CSF) can beadministered to the subject, e.g., to boost the immune response.

The term antibody as used herein refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules (i.e.,molecules that contain an antigen binding site that specifically bindsto the peptide (e.g., a peptide described herein)). An antibody thatspecifically binds to a peptide described herein is an antibody thatbinds the peptide, but does not substantially bind other molecules in asample. Examples of immunologically active portions of immunoglobulinmolecules include, e.g., F(ab) fragments, F(ab′)2 fragments, or anyother antibody fragments described herein.

An isolated antibody or antigen-binding fragment thereof produced by themethods described herein can selectively bind to an epitope within oroverlapping the amino acid sequence of any of SEQ ID NOs: 1-17. Theantibody can also be one that cross-blocks the binding of antibody thatbinds to an epitope within or overlapping the amino acid sequence of anyof SEQ ID NOs: 1-17. Typically, binding of an antibody to an epitope isconsidered selective when the antibody binds with a KD of less than 10⁻⁶M. If necessary, nonspecific binding can be reduced withoutsubstantially affecting selective binding by varying the bindingconditions. An antibody that “crossblocks” or a “crossblocking antibody”refers to a first antibody that, when bound to an epitope (e.g., onecontained within or overlapping any of SEQ ID NOs: 1-17), reduces oreliminates the ability of a second antibody to bind to the peptide(relative to binding of the second antibody to the peptide that occursin the absence of the first antibody). It is understood that an antibodyproduced by a method described herein (e.g., an antibody specific forone or more of the peptides described herein) can be used to, e.g.,detect a cancer cell expressing BCMA or TACI and thus is useful in manyexemplary methods described herein.

Immunological Testing

The antigen-specific cellular immune responses of vaccinated subjectscan be monitored by a number of different assays, such as tetramerassays, ELISPOT, and quantitative PCR. These methods and protocols aredescribed, e.g., in Current Protocols in Immunology, Coligan, J. et al.,Eds., (John Wiley & Sons, Inc.; New York, N.Y.).

A tetramer assay can be used to detect and quantify T-cells that arespecific for a given antigen within a blood sample. Tetramers comprisedof recombinant MHC molecules complexed with peptide can be used toidentify populations of antigen-specific T cells. To detect T cellsspecific for antigens, fluorochrome labeled specific peptide tetramercomplexes (e.g., phycoerythrin (PE)-tHLA) containing peptides from theseantigens are synthesized and provided by Beckman Coulter (San Diego,Calif.). Specific CTL clone CD8 cells are resuspended at 10⁵ cells/50 μlFACS buffer (phosphate buffer plus 1% inactivated FCS buffer). Cells areincubated with 1 μl tHLA for 30 minutes at room temperature andincubation is continued for 30 minutes at 4° C. with 10 μl anti-CD8 mAb(Becton Dickinson, San Jose, Calif.). Cells are washed twice in 2 mlcold FACS buffer before analysis by FACS (Becton Dickinson).

ELISPOT assays can be used to detect cytokine secreting cells, e.g., todetermine whether cells in a vaccinated patient secrete cytokine inresponse to antigen, thereby demonstrating whether antigen-specificresponses have been elicited. ELISPOT assay kits are supplied from R & DSystems (Minneapolis, Minn.) and performed as described by themanufacturer's instructions. Responder (R) 1×10⁵ patients' PBMC cellsfrom before and after vaccination are plated in 96-well plates withnitrocellulose membrane inserts coated with capture Ab. Stimulator (S)cells (TAP-deficient T2 cells pulsed with antigen) are added at the R:Sratio of 1:1. After a 24-hour incubation, cells are removed by washingthe plates 4 times. The detection Ab is added to each well. The platesare incubated at 4° C. overnight and the washing steps will be repeated.After a 2-hour incubation with streptavidin-AP, the plates are washed.Aliquots (100 μl) of BCIP/NBT chromogen are added to each well todevelop the spots. The reaction is stopped after 60 min by washing withwater. The spots are scanned and counted with computer-assisted imageanalysis (Cellular Technology Ltd, Cleveland, Ohio). When experimentalvalues are significantly different from the mean number of spots againstnon-pulsed T2 cells (background values), as determined by a two-tailedWilcoxon rank sum test, the background values are subtracted from theexperimental values.

Quantitative PCR is another means for evaluating immune responses. Toexamine IFN-γ production in patients by quantitative PCR, cryopreservedPBMCs from patients' pre-vaccination and post-vaccinations samples andautologous dendritic cells are thawed in RPMI DC culture medium with 10%patient serum, washed and counted. PBMC are plated at 3×10⁶ PBMCs in 2ml of medium in 24-well plate; dendritic cells are plated at 1×10⁶/mland are pulsed 24 hour with 10 μg/ml tumor peptide in 2 ml in each wellin 24 well plate. Dendritic cells are collected, washed, and counted,and diluted to 1×10⁶/ml, and 3×10⁵ (i.e., 300 μl solution) added towells with PBMC (DC:PBMC=1:10). 2.3 μl IL-2 (300 IU/mL) is added every3-4 days, and the cells are harvested between day 10 and day 13 afterinitiation of the culture. The harvested cells are then stimulated withtumor cells or autologous PBMC pulsed with 10 μg/ml tumor peptide for 4hours at 37° C. On days 11-13, cultures are harvested, washed twice,then divided into four different wells, two wells using for control(without target); and another two wells CTL cocultured with tumor cells(1:1) if tumor cells are available. If tumor cells are not available, 10μg/ml tumor lysate is added to CTL. After 4 hours of stimulation, thecells are collected, RNA extracted, and IFN-γ and CD8 mRNA expressionevaluated with a thermocycler/fluorescence camera system. PCRamplification efficiency follows natural log progression, with linearregression analyses demonstrating correlation co-efficients in excess of0.99. Based on empirical analysis, a one-cycle difference is interpretedto be a two-fold difference in mRNA quantity, and CD8-normalized IFN-γquantities are determined. An increase of >1.5-fold in post-vaccinerelative to pre-vaccine IFN-γ is the established standard for positivetype I vaccine responsiveness.

Methods for Selecting a Therapy

Methods for selecting a therapy for a subject with a cancer (e.g., aplasma cell disorder such as multiple myeloma or any cancer in whichBCMA or TACI are expressed or overexpressed) include the steps of:optionally, determining whether one or more cancer cells of the subjectexpress or over express BCMA or TACI; and if one or more cells expressBCMA or TACI, selecting as a therapy for the subject a compositioncontaining at least one peptide as described herein (a peptidecomprising the amino acid sequence of any one of SEQ ID NOs: 1-17, apeptide comprising the amino acid sequence that is at least 50%, 60%,70%, 80%, or 90% identical to SEQ ID NOs: 1-17, or have no more than 4substitutions of, the amino acid sequence of any of SEQ ID NOS: 1-17),provided that the amino acid sequence is capable of: (i) inducing in thesubject an immune response; (ii) binding to an MHC molecule; and (iii)being recognized, in the context of an MHC molecule, by a T cellreceptor on a T cell.

In some embodiments, the methods further include the steps of determinewhether one or more cancer cells of the subject express a MHC molecule,e.g., an MHC class I molecule (e.g., HLA-A2), or an MHC class IImolecule.

It is understood that where one or more cells (e.g., plasma cells) of asubject's cancer express or overexpress both BCMA and TACI, acombination of suitable peptides can be delivered to the subject. Forexample, where one or more cells (e.g., plasma cells) of a subject'scancer are determined to express or overexpress both BCMA and TACI, themethods for selecting a therapy can include selecting as a therapy forthe subject: a combination of a composition containing at least onepeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1-6and 13-14, and a composition containing at least one peptide comprisingthe amino acid sequence of any one of SEQ ID NOs: 7-12 and 15-17.

Methods for determining whether one or more cells express BCMA, TACI,and/or a MHC molecule are known in the art. For example, a biologicalsample (e.g., a blood sample or lymph node tissue sample) obtained froma subject can be tested using an BCMA and/or TACI-specific antibody madeby a method described herein to detect the presence or amount of an BCMAand TACI polypeptide expressed by a cell (or cell lysate). Methods forassaying a biological sample for the presence or amount of a polypeptideinclude, e.g., ELISA, immunohistochemistry, flow cytometry,western-blotting, or dot-blotting assays. In some embodiments, any ofthe methods described herein can also include the step of providing abiological sample from a subject and/or obtaining a biological samplefrom a subject. Suitable biological samples for the methods describedherein include any biological fluid, cell, tissue, or fraction thereof,which includes analyte proteins of interest. A biological sample can be,for example, a specimen obtained from a subject or can be derived fromsuch a subject. For example, a sample can be a tissue section obtainedby biopsy, or cells that are placed in or adapted to tissue culture. Abiological sample can also be a cell-containing biological fluid such asurine, blood, plasma, serum, saliva, semen, sputum, cerebral spinalfluid, tears, mucus or an aspirate (e.g., a lung or breast nippleaspirate), or such a sample absorbed onto a paper or polymer substrate.A biological sample can be further fractionated, if desired, to afraction containing particular cell types. For example, a blood samplecan be fractionated into serum or into fractions containing particulartypes of blood cells such as red blood cells or white blood cells(leukocytes). If desired, a sample can be a combination of sample typesfrom a subject such as a combination of a tissue and biological fluid.

The biological samples can be obtained from a subject, e.g., a subjecthaving, suspected of having, or at risk of developing, a cancer (e.g.,multiple myeloma). Any suitable methods for obtaining the biologicalsamples can be employed, although exemplary methods include, e.g.,phlebotomy, swab (e.g., buccal swab), aspiration, or fine needleaspirate biopsy procedure. Non-limiting examples of tissues susceptibleto fine needle aspiration include lymph node, lung, thyroid, breast, andliver. Samples can also be collected, e.g., by microdissection (e.g.,laser capture microdissection (LCM) or laser microdissection (LMD)),bladder wash, smear (PAP smear), or ductal lavage.

A medical practitioner can also select, prescribe and/or administer oneor more additional therapeutic agents to treat a cancer or one or moremedicaments to treat side-effects of an anti-cancer agent. Suitablechemotherapeutic agents for treating multiple myeloma include, e.g.,melphalan, cyclophosphamide, vincristine, doxorubicin, prednisone,dexamethasone, proteasome inhibitors (e.g., bortezomib), thalidomide, orlenalidomide. Side effects of anti-cancer agents include, e.g., anemia,gastrointestinal symptoms (e.g., nausea, vomiting, diarrhea), leukopenia(decreased number of white blood cells, which may cause infection),temporary hair loss, or thrombocytopenia (decreased number of platelets,which may cause bleeding). Thus, a medical practitioner can prescribe oradminister to a subject a chemotherapeutic agent such as vincristinealong with an anti-anemia medicament such as epoetin alpha (e.g.,Procrit® or Epogen®).

Nucleic Acid Vaccines

The present disclosure provides Nucleic Acid Vaccines (NAVs) comprisingone or more polynucleotides, e.g., polynucleotide constructs, whichencode one or more polypeptides as described herein. Exemplarypolynucleotides include e.g., polynucleotide constructs, include DNA,RNA, antigen-encoding RNA polynucleotides, e.g., mRNAs. In someembodiments, the polynucleotides, e.g., antigen-encoding RNApolynucleotides, can include at least one chemical modification. In someembodiments, the nucleic acid vaccines can be formulated within apolymeric or liposomal nanocarrier (e.g., a nanoparticle).

In some embodiments, adjuvants or immune potentiators, can also beadministered with or in combination with one or more NAVs. In someembodiments, an adjuvant acts as a co-signal to prime T-cells and/orB-cells and/or NK cells.

NAVs can vary in their valency. Valency refers to the number ofantigenic components in the NAV or NAV polynucleotide (e.g., RNApolynucleotide) or polypeptide. In some embodiments, the NAVs aremonovalent. In some embodiments, the NAVs are divalent. In someembodiments, the NAVs are trivalent. In some embodiments the NAVs aremulti-valent. Multivalent vaccines can comprise 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more antigens orantigenic moieties (e.g., antigenic peptides, etc.). The antigeniccomponents of the NAVs can be on a single polynucleotide or on separatepolynucleotides.

The NAVs can be used as therapeutic or prophylactic agents. They areprovided for use in medicine and/or for the priming of immune effectorcells, e.g., stimulate/transfect peripheral blood mononuclear cells(PBMCs) ex vivo and re-infuse the activated cells. For example, a NAVdescribed herein can be administered to a subject, wherein thepolynucleotides is translated in vivo to produce an antigen. Providedare compositions, methods, kits, and reagents for diagnosis, treatmentor prevention of a disease or condition in humans and other mammals. Theactive therapeutic agents can include NAVs, cells containing NAVs orpolypeptides translated from the polynucleotides contained in said NAVs.

Provided herein are methods of inducing translation of a polypeptide(e.g., antigen or immunogen) in a cell, tissue or organism using thepolynucleotides of the NAVs described herein. Such translation can be invivo, ex vivo, in culture, or in vitro. The cell, tissue or organism iscontacted with an effective amount of a composition containing a NAVwhich contains a polynucleotide that has at least one a translatableregion encoding the polypeptide of interested (e.g., antigen orimmunogen).

An “effective amount” of the NAV composition is provided based, at leastin part, on the target tissue, target cell type, means ofadministration, physical characteristics of the polynucleotide (e.g.,size, and extent of modified nucleosides) and other components of theNAV, and other determinants. In general, an effective amount of the NAVcomposition provides an induced or boosted immune response as a functionof antigen production in the cell, preferably more efficient than acomposition containing a corresponding unmodified polynucleotideencoding the same antigen. Increased antigen production can bedemonstrated by increased cell transfection (i.e., the percentage ofcells transfected with the NAV), increased protein translation from thepolynucleotide, decreased nucleic acid degradation (as demonstrated,e.g., by increased duration of protein translation from a modifiedpolynucleotide), or altered innate immune response of the host cell.

The present disclosure also provides methods of inducing in vivotranslation of a polypeptide antigen in a mammalian subject in needthereof. Therein, an effective amount of a NAV composition containing apolynucleotide that has at least one structural or chemical modificationand a translatable region encoding the polypeptide (e.g., antigen orimmunogen) is administered to the subject using the delivery methodsdescribed herein. The polynucleotide is provided in an amount and underother conditions such that the polynucleotide is translated in the cell.The cell in which the polynucleotide is localized, or the tissue inwhich the cell is present, can be targeted with one or more than onerounds of NAV administration.

The proteins described herein can be engineered for localization withinthe cell, potentially within a specific compartment such as thecytoplasms or nucleus, or are engineered for secretion from the cell ortranslocation to the plasma membrane of the cell.

In some embodiments, the nucleic acid (e.g., DNA, RNA) can have one ormore modifications. In some embodiments, the nucleic acid molecule(e.g., an RNA molecule) as defined herein can contain nucleotideanalogues/modifications, e.g. backbone modifications, sugarmodifications or base modifications. A backbone modification inconnection with the present invention is a modification, in whichphosphates of the backbone of the nucleotides contained in a nucleicacid molecule as defined herein are chemically modified. A sugarmodification in connection with the present invention is a chemicalmodification of the sugar of the nucleotides of the nucleic acidmolecule as defined herein. Furthermore, a base modification inconnection with the present invention is a chemical modification of thebase moiety of the nucleotides of the nucleic acid molecule of thenucleic acid molecule. In this context, nucleotide analogues ormodifications are preferably selected from nucleotide analogues whichare applicable for transcription and/or translation.

The modified nucleosides and nucleotides, which can be incorporated intothe nucleic acid molecule can be modified in the sugar moiety. Forexample, the 2′ hydroxyl group (OH) of an RNA molecule can be modifiedor replaced with a number of different “oxy” or “deoxy” substituents.Examples of “oxy”-2′ hydroxyl group modifications include, but are notlimited to, alkoxy or aryloxy (—OR, e.g., R=H, alkyl, cycloalkyl, aryl,aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG),—O(CH₂CH₂O)nCH₂CH₂OR; “locked” nucleic acids (LNA) in which the 2′hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon ofthe same ribose sugar; and amino groups (—O-amino, wherein the aminogroup, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl,arylamino, diarylamino, heteroarylamino, or diheteroaryl amino, ethylenediamine, polyamino) or aminoalkoxy.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, a modified nucleic acid molecule can includenucleotides containing, for instance, arabinose as the sugar.

The phosphate backbone can further be modified in the modifiednucleosides and nucleotides, which can be incorporated into the nucleicacid molecule (e.g., an RNA) as described herein. The phosphate groupsof the backbone can be modified by replacing one or more of the oxygenatoms with a different substituent. Further, the modified nucleosidesand nucleotides can include the full replacement of an unmodifiedphosphate moiety with a modified phosphate as described herein. Examplesof modified phosphate groups include, but are not limited to,phosphorothioate, phosphoroselenoates, borano phosphates, boranophosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl oraryl phosphonates and phosphotriesters. Phosphorodithioates have bothnon-linking oxygens replaced by sulfur. The phosphate linker can also bemodified by the replacement of a linking oxygen with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylene-phosphonates).

The modified nucleosides and nucleotides, which can be incorporated intothe nucleic acid molecule (e.g., an RNA molecule) as described herein,can further be modified in the nucleobase moiety. Examples ofnucleobases found in RNA include, but are not limited to, adenine,guanine, cytosine and uracil. For example, the nucleosides andnucleotides described herein can be chemically modified on the majorgroove face. In some embodiments, the major groove chemicalmodifications can include an amino group, a thiol group, an alkyl group,or a halo group.

In some embodiments, the nucleotide analogues/modifications are selectedfrom base modifications, which can be selected, e.g., from2-amino-6-chloropurineriboside-5′-triphosphate,2-Aminopurine-riboside-5′-triphosphate;2-aminoadenosine-5′-triphosphate,2′-Amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate,2′-O-Methyl inosine-5′-triphosphate 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate,5-Bromo-2′-deoxycytidine-5′-triphosphate,5-Bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-Iodo-2′-deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-Iodo-2′-deoxyuridine-5′-triphosphate,5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate,5-Propynyl-2′-deoxycytidine-5′-triphosphate,5-Propynyl-2′-deoxyuridine-5′-triphosphate,6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate,6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

In some embodiments, the nucleic acid molecule can be modified by theaddition of a so-called “5′-CAP” structure. A 5′-cap is an entity,typically a modified nucleotide entity, which generally “caps” the5′-end of a mature mRNA. A 5′-cap can typically be formed by a modifiednucleotide, particularly by a derivative of a guanine nucleotide.Preferably, the 5′-cap is linked to the 5′-terminus via a5′-5′-triphosphate linkage. A 5′-cap can be methylated, e.g. m7GpppN,wherein N is the terminal 5′ nucleotide of the nucleic acid carrying the5′-cap, typically the 5′-end of an RNA. m7GpppN is the 5′-CAP structurewhich naturally occurs in mRNA transcribed by polymerase II and istherefore not considered as modification comprised in the modified RNAaccording to the invention.

How to make and use nucleic acid vaccines are described, e.g., inUS20070269451, US20160317647, U.S. Pat. No. 9,872,900, and US2017002984each of which is incorporated herein by reference in its entirety.

Pharmaceutical Compositions

Any of the peptides, nucleic acids encoding the peptides, nanoparticles,and cells described herein can be incorporated into pharmaceuticalcompositions. The compositions can include one or more of the peptides(and/or nucleic acids encoding the peptides) and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” includes solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Oneor more peptides can be formulated as a pharmaceutical composition inthe form of a syrup, an elixir, a suspension, a powder, a granule, atablet, a capsule, a lozenge, a troche, an aqueous solution, a cream, anointment, a lotion, a gel, an emulsion, etc. Supplementary activecompounds (e.g., one or more chemotherapeutic agents) can also beincorporated into the compositions.

A pharmaceutical composition is generally formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration include oral, rectal, and parenteral, e.g., intravenous,intramuscular, intradermal, subcutaneous, inhalation, transdermal, ortransmucosal. Solutions or suspensions used for parenteral applicationcan include the following components: a sterile diluent such as waterfor injection, saline solution, fixed oils, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide. The compositions can be enclosedin ampoules, disposable syringes or multiple dose vials made of glass orplastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the pharmaceutical composition must be sterile and should befluid to the extent that easy syringability exists. It should be stableunder the conditions of manufacture and storage and must be preservedagainst any contamination by microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of contamination by microorganisms can beachieved by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and thelike. In many cases, it will be desirable to include isotonic agents,for example, sugars, polyalcohols such as mannitol, sorbitol, sodiumchloride in the composition. Prolonged absorption of the injectablecompositions can be facilitated by including in the composition an agentthat delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating one ormore of the peptides (or one or more the nucleic acids encoding thepeptides) in the required amount in an appropriate solvent with one or acombination of ingredients, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thepeptide(s) (or nucleic acid(s) encoding the peptide(s)) into a sterilevehicle which contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the methods ofpreparation can include vacuum drying or freeze-drying which yields apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the one ormore peptides can be incorporated with excipients and used in the formof tablets, troches, or capsules, e.g., gelatin capsules. Oralcompositions can also be prepared using a fluid carrier for use as amouthwash. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orsterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

The powders and tablets can contain from 1% to 95% (w/w) of anindividual peptide or a mixture of two or more peptides. In certainembodiments, the peptide can range from about 5% to 70% (w/w). Suitablecarriers are magnesium carbonate, magnesium stearate, talc, sugar,lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose,sodium carboxymethylcellulose, a low melting wax, cocoa butter, and thelike. The term “preparation” is intended to include the formulation ofthe peptide (or nucleic acid) with encapsulating material as a carrierproviding a capsule in which the peptide with or without other carriers,is surrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid dosage formssuitable for oral administration.

Aqueous solutions suitable for oral use can be prepared by dissolvingthe active component in water and adding suitable colorants, flavors,stabilizers, and thickening agents as desired. Aqueous suspensionssuitable for oral use can be made by dispersing the finely dividedactive component in water with viscous material, such as natural orsynthetic gums, resins, methylcellulose, sodium carboxymethylcellulose,and other well-known suspending agents.

For administration by inhalation, the peptides or nucleic acids can bedelivered in the form of an aerosol spray from pressured container ordispenser which contains a suitable propellant, e.g., a gas such ascarbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the peptides or nucleic acids can beformulated into ointments, salves, gels, or creams as generally known inthe art.

The peptides or nucleic acids can also be prepared in the form ofsuppositories (e.g., with conventional suppository bases such as cocoabutter and other glycerides) or retention enemas for rectal delivery.

In some embodiments, the peptides or nucleic acids can be prepared withcarriers that will protect the peptides against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to, e.g., APCs with monoclonal antibodiesto APC-specific antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It can be advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form, as used herein, refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of the peptides (or nucleic acids)calculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. Dosage units can also beaccompanied by instructions for use.

The nucleic acid molecules encoding the peptides can be inserted intovectors and used as gene therapy vectors. Gene therapy vectors can bedelivered to a subject by, for example, intravenous injection, localadministration (see, e.g., U.S. Pat. No. 5,328,470) or by stereotacticinjection (see, e.g., Chen, et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

Additional examples of gene delivery vehicles include, but are notlimited to, liposomes, biocompatible polymers, including naturalpolymers and synthetic polymers; lipoproteins; polypeptides;polysaccharides; lipopolysaccharides; artificial viral envelopes; metalparticles; bacteria; viruses such as baculovirus, adenovirus, andretrovirus; bacteriophage; cosmids; plasmids; fungal vectors and otherrecombination vehicles typically used in the art which have beendescribed for expression in a variety of eukaryotic and prokaryotichosts, and may be used for gene therapy as well as for simple proteinexpression.

Examples of viral vectors include retroviral vectors, lentivirusvectors, adenovirus vectors, adeno-associated virus vectors, alphavirusvectors and the like. Liposomes that comprise a targeting moiety such asan antibody or fragment thereof can also be used to preparepharmaceutical compositions of nucleic acids for delivery to a subject.

Any of the pharmaceutical compositions described herein can be includedin a container, pack, or dispenser together with instructions foradministration as described below.

Kits and Articles of Manufacture

The disclosure also features a variety of kits. The kits can include,e.g., one or more (e.g., one, two, three, four, five, six, seven, eight,nine, or 10 or more) of any of the peptides (or expression vectorscontaining nucleic acid sequences encoding one or more peptides)described herein; and instructions for administering the peptide to asubject. The kit can include one or more pharmaceutically acceptablecarriers and/or one or more immune stimulating agents. The immunestimulating agents can be, e.g., a T helper epitope, an altered peptideligand, or an adjuvant. The kits can also contain one or moretherapeutic agents, diagnostic agents, or prophylactic agents. The oneor more therapeutic, diagnostic, or prophylactic agents include, but arenot limited to: (i) an agent that modulates inflammatory responses(e.g., aspirin, indomethacin, ibuprofen, naproxen, steroids, cromolynsodium, or theophylline); (ii) an agent that affects renal and/orcardiovascular function (e.g., furosemide, thiazide, amiloride,spironolactone, captopril, enalapril, lisinopril, diltiazem, nifedipine,verapamil, digoxin, isordil, dobutamine, lidocaine, quinidine,adenosine, digitalis, mevastatin, lovastatin, simvastatin, ormevalonate); (iii) drugs that affect gastrointestinal function (e.g.,omeprazole or sucralfate); (iv) antibiotics (e.g., tetracycline,clindamycin, amphotericin B, quinine, methicillin, vancomycin,penicillin G, amoxicillin, gentamicin, erythromycin, ciprofloxacin,doxycycline, streptomycin, gentamicin, tobramycin, chloramphenicol,isoniazid, fluconazole, or amantadine); (v) anti-cancer agents (e.g.,cyclophosphamide, methotrexate, fluorouracil, cytarabine,mercaptopurine, vinblastine, vincristine, doxorubicin, bleomycin,mitomycin C, hydroxyurea, prednisone, tamoxifen, cisplatin, ordacarbazine); (vi) immunomodulatory agents (e.g., interleukins,interferons (e.g., interferon gamma (IFN-γ), granulocytemacrophage-colony stimulating factor (GM-CSF), tumor necrosis factoralpha (TNFα), tumor necrosis factor beta (TNFβ), cyclosporine, FK506,azathioprine, steroids); (ix) drugs acting on the blood and/or theblood-forming organs (e.g., interleukins, G-CSF, GM-CSF, erythropoietin,heparin, warfarin, or coumarin); or (vii) hormones (e.g., growth hormone(GH), prolactin, luteinizing hormone, TSH, ACTH, insulin, FSH, CG,somatostatin, estrogens, androgens, progesterone, gonadotropin-releasinghormone (GnRH), thyroxine, triiodothyronine); hormone antagonists;agents affecting calcification and bone turnover (e.g., calcium,phosphate, parathyroid hormone (PTH), vitamin D, bisphospho nates,calcitonin, fluoride).

In some embodiments, the kits can contain one or more (e.g., one, two,or three or more) of any of the BCMA and/or TACI antibodies describedherein. In some embodiments, the kits can include two antibodies, eachspecific for a different protein. For example, a kit can contain oneBCMA-specific antibody (described herein) and one TACI-specific antibody(described herein). The kits can optionally include instructions forassaying a biological sample for the presence or amount of one or moreof BCMA, and/or TACI proteins. Also featured are articles of manufacturethat include: a container; and a composition contained within thecontainer, wherein the composition comprises an active ingredient forinducing an immune response in a mammal (e.g., a human), wherein theactive ingredient comprises one or more (e.g., two, three, four, five,six, seven, eight, nine, or 10 or more) of any of the peptides describedherein, and wherein the container has a label indicating that thecomposition is for use in inducing an immune response in a mammal (e.g.,any of the mammals described herein). The label can further indicatethat the composition is to be administered to a mammal having, suspectedof having, or at risk of developing, multiple myeloma. The compositionof the article of manufacture can be dried or lyophilized and caninclude, e.g., one or more solutions (and/or instructions) forsolubilizing a dried or lyophilized composition.

The articles of manufacture can also include instructions foradministering the composition to the mammal.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1. BCMA Expression on Multiple Myeloma Cell Lines

A total of 12 cancer cell lines including 11 MM cell lines and 1 breastcancer cell line (MDA-MB231) were evaluated for their expression levelsof BCMA antigen by staining with an antibody specific to each followingclone; #1. ANC3B1 (LifeSpan Biosciences, Cat #LS-C357630), #2. VICKY1(LifeSpan Biosciences, Cat #LS-C18662), and #3. 19F2 (BioLegend, Cat#357506). Among the cell lines, H929 (MM cell line) showed the highestlevel of BCMA expression and MDA-MB231 (breast cancer cell line; BCMAnegative) showed the minimum level of BCMA expression. (FIGS. 1A-1I).

Example 2. Selection of BCMA and TACI Native Peptides Specific to HLA-A2

Six native peptides derived from BCMA or TACI antigen, respectively,were identified as following:

#1. BCMA₆₄₋₇₂ (SEQ ID NO: 1) (LIISLAVFV) #2. BCMA₆₉₋₇₇ (SEQ ID NO: 2)(AVFVLMFLL) #3. BCMA₉₋₁₇ (SEQ ID NO: 3) (SQNEYFDSL) #4. BCMA₇₂₋₈₀(SEQ ID NO: 4) (VLMFLLRKI) #5. BCMA₅₄₋₆₂ (SEQ ID NO: 5) (AILWTCLGL)#6. BCMA₁₁₄₋₁₂₂ (SEQ ID NO: 6) (ILPRGLEYT) #1. TACI₁₇₈₋₁₈₆(SEQ ID NO: 7) (FLVAVACFL) #2. TACI₁₇₄₋₁₈₂ (SEQ ID NO: 8) (VLCCFLVAV)#3. TACI₁₅₄₋₁₆₂ (SEQ ID NO: 9) (KLSADQVAL) #4. TACI₁₆₆₋₁₇₄(SEQ ID NO: 10) (TLGLCLCAV) #5. TACI₁₆₁₋₁₆₉ (SEQ ID NO: 11) (ALVYSTLGL)#6. TACI₁₅₅₋₁₆₃ (SEQ ID NO: 12) (LSADQVALV)

Example 3. Binding Affinity of BCMA or TACI Native Peptides to HLA-A2Molecule

The listed BCMA peptides were evaluated for HLA-A2-specific bindingcapacity using the T2 cell line. In the assay, T2 cells were washed,resuspended in serum-free AIM-V medium to a final concentration of 1×10⁶cells/ml and transferred into wells of a 24-well tissue culture plate.The cells were pulsed with different concentrations of respective BCMApeptide (0-200 μg/ml) plus 3 μg/ml human β2-microglobulin (Sigma) andincubated at 37° C., 5% CO₂ in humidified air. Following overnightincubation, the cells were washed, stained with mouse anti-humanHLA-A2-FITC mAb for 15 minutes at 4° C., washed and analyzed using aFACSort™ flow cytometer with CellQuest™ v2.1 software (Becton Dickinson,San Jose, Calif.). Peptide binding to HLA-A2 was determined by theup-regulation of HLA-A2 molecules on T2 cells caused by HLA-A2 specificpeptide binding and demonstrated by measuring mean fluorescenceintensity (MFI) by flow cytometric analyses. Among the BCMA peptidesevaluated, “#4. BCMA₇₂₋₈₀ (VLMFLLRKI (SEQ ID NO: 4))” showed the highestlevel of HLA-A2 specificity and “#5. BCMA₅₄₋₆₂ (AILWTCLGL (SEQ ID NO:5))” showed the second highest level of the specificity. (FIG. 2 ).Among the TACI peptides evaluated, all peptides expect for TACI #5showed HLA-A2 specificity, but the highest level was measured by #4.TACI₁₆₆₋₁₇₄ (TLGLCLCAV (SEQ ID NO: 10)) (FIG. 3 ).

Example 4. Stability of BCMA or TACI Native Peptides to HLA-A2 Molecule

In order to improve the stability of the peptide binding to HLA-A2molecules, the following heteroclitic BCMA or TACI peptides weredesigned:

Heteroclitic #4. BCMA₇₂₋₈₀ (SEQ ID NO: 13) (YLMFLLRKI)Heteroclitic #5. BCMA₅₄₋₆₂ (SEQ ID NO: 14) (YILWTCLGL)Heteroclitic #1. TACI₁₇₈₋₁₈₆ (SEQ ID NO: 15) (YLVAVACFL)Heteroclitic #3. TACI₁₅₄₋₁₆₂ (SEQ ID NO: 16) (YLSADQVAL)Heteroclitic #4. TACI₁₆₆₋₁₇₄ (SEQ ID NO: 17) (YLGLCLCAV)

The native and heteroclitic BCMA and TACI peptides were examined forHLA-A2 binding stability using the T2 cell line. T2 cells were pulsedwith the respective peptide. After overnight incubation, the cells werewashed to remove unbound peptide; they were evaluated for bindingaffinity as shown above and stability as following. The cells wereincubated with 10 μg/ml Brefeldin A (Sigma) at 37° C. and 5% CO₂ for 1hour to block cell surface expression of newly synthesized HLA-A2molecules. Peptide/HLA-A2 binding stability was evaluated at 0, 2, 4, 6and 18 hours post-Brefeldin A treatment. Following the incubationperiod, the cells were harvested, washed, stained with mouse anti-humanHLA-A2-FITC mAb and analyzed by flow cytometry. The HLA-A2 bindingaffinity of the “Heteroclitic #4 BCMA₇₂₋₈₀ (YLMFLLRKI (SEQ ID NO: 13))”,“Heteroclitic #5 BCMA₅₄₋₆₂ (YILWTCLGL (SEQ ID NO: 14))” and“Heteroclitic #3 TACI₁₅₄₋₁₆₂ (YLSADQVAL (SEQ ID NO: 16))” was increasedfrom their native peptide (FIGS. 4 and 5 ). In terms of the bindingstability, “Heteroclitic #4 BCMA₇₂₋₈₀. (YLMFLLRKI (SEQ ID NO: 13))” and“Heteroclitic #3 TACI₁₅₄₋₁₆₂ (YLSADQVAL (SEQ ID NO: 16))” peptidesshowed a significant improvement in their HLA-A2 affinity at all thetime points evaluated including 0, 2, 4, 6 and 18 hours compared to thenative peptide (FIGS. 6 and 7 ). Therefore, the Heteroclitic #4BCMA₇₂₋₈₀ (YLMFLLRKI (SEQ ID NO: 13)) and Heteroclitic #3. TACI₁₅₄₋₁₆₂(YLSADQVAL (SEQ ID NO: 16) peptides were selected for further evaluationof their immunogenic potential to generate MM-specific cytotoxic T cells(CTLs).

Example 5. Induction of BCMA or TACI Peptide-Specific CD3⁺CD8⁺ CTL

The peptide-specific CTL were generated from different HLA-A2⁺ normaldonors for the evaluation of the functional activities targeting MM celllines. To generate the peptide-specific CTL, mature dendritic cells(mDC) generated from the same donor were resuspended in serum-free AIM-Vmedia and pulsed with 50 μg/ml of the Heteroclitic #4 BCMA₇₂₋₈₀(YLMFLLRKI (SEQ ID NO: 13)) peptide or “Heteroclitic #3 TACI₁₅₄₋₁₆₂(YLSADQVAL (SEQ ID NO: 16))” peptide, overnight at 37° C., 5% CO₂ inhumidified air. The peptide-pulsed mDC were washed, counted, irradiatedat 10 Gy and used to prime CD3⁺ T cells at a 1:20 antigen-presentingcells/peptide-to-CD3⁺ T cell ratio in AIM-V media supplemented with 10%human AB serum. The cultures were restimulated every seven days withirradiated T2 cells pulsed with peptide for a total of 4 cycles. Tomaintain the T cells ex vivo, IL-2 (50 U/ml) was added to the culturestwo days after the second stimulation. Control T cell cultures weremaintained under the same culture conditions in the presence of IL-2 (50U/ml), but without peptide stimulation. Phenotype of the resulting CTLwas evaluated one week after each cycle of peptide stimulation. Flowcytometric analysis showed a distinct change in the phenotype of theCD3⁺CD8⁺ T cell subsets stimulated with the Heteroclitic #4 BCMA₇₂₋₈₀(YLMFLLRKI (SEQ ID NO: 13)) with a gradual increase in the population.The CD3⁺CD8⁺ T cell increases by the heteroclitic BCMA peptide wassimilar to those with the immunogenic CD138₂₆₀₋₂₆₈ (GLVGLIFAV (SEQ IDNO: 20)), which was previously identified as immunogenic peptide,suggesting the potential immunogenicity of the BCMA peptide. The BCMApeptide-specific CTL cultures contained a higher percentage of CD8⁺ Tcells (˜80%) upon 4 cycle of peptide stimulation compared to non-peptidestimulated control T cells (˜20%) (FIGS. 8A-8C).

Example 6. Decreased Naïve and Increased Memory CD3⁺CD8⁺ CTL byHeteroclitic BCMA₇₂₋₈₀ Peptide Stimulation

Antigen-specific CTL can be phenotypically identified asactivated/memory T cells from naïve T cells by their expression ofdistinct cell surface antigens. The phenotype of the BCMA-CTL wereexamined as potential effector cells by analyzing the phenotype of naïveand memory cells. BCMA peptide-specific CTL were generated by repeatedstimulation of HLA-A2⁺ normal donor's CD3⁺ T cells weekly withantigen-presenting cells pulsed with 50 μg/ml heteroclitic BCMA₇₂₋₈₀(YLMFLLRKI (SEQ ID NO: 13)). One week after each peptide stimulation,the resulting CTL were evaluated for their phenotypic profile by flowcytometry. The BCMA-CTL showed a decreased frequency of naive CD3⁺CD8⁺ Tcells as compared to the control T cells (Donor 1: 80% unstimulated to2% upon 4 cycles of stimulation; Donor 2: 83% unstimulated to 2% upon 4cycles of stimulation). A corresponding increase was observed in thefrequency of the memory CD3⁺CD8⁺ T cells (Donor 1: 18% unstimulated to86% upon 4 cycles of stimulation; Donor 2: 10% unstimulated to 92% upon4 cycles of stimulation) with the heteroclitic BCMA₇₂₋₈₀ (YLMFLLRKI (SEQID NO: 13)) peptide. These phenotypic changes demonstrate that repeatedstimulation of CD3⁺ T cells with heteroclitic BCMA₇₂₋₈₀ (YLMFLLRKI (SEQID NO: 13)) resulted in an expansion of CD8⁺ CTL with a phenotype ofmemory cells, indicating the immunogenicity of the BCMA peptide (FIGS. 9and 10 ).

Example 7. Changes in Frequency of Central Memory and Effector CD3⁺CD8⁺CTL by Heteroclitic BCMA₇₂₋₈₀ Peptide Stimulation

Further evaluation of central memory and effector cells was performed,upon the stimulation of T cells with heteroclitic BCMA₇₂₋₈₀ (YLMFLLRKI(SEQ ID NO: 13)) peptide. The expansion of central memory CTL by theBCMA peptide was detected after 3 cycle of stimulation, which wasaligned with a decrease of effector CTL. Upon 4 cycle of the peptidestimulation, a decrease in central memory CTL and increase in effectorCTL including effector memory cells were also detected. The pattern ofthis phenotype change in the CD8+ T cells with the heterocliticBCMA₇₂₋₈₀ (YLMFLLRKI (SEQ ID NO: 13)) peptide was similar to the cellsstimulated with CD138₂₆₀₋₂₆₈ (GLVGLIFAV (SEQ ID NO: 20)) (FIGS.11A-11C).

Example 8. The Specific CTL Stimulated with Heteroclitic BCMA₇₂₋₈₀(YLMFLLRKI—SEQ ID NO: 13) Peptide or Heteroclitic TACI₁₅₄₋₁₆₂(YLSADQVAL—SEQ ID NO: 16) Peptide Display a Distinct PhenotypeRepresenting Specific T Cell Subtypes

We also observed distinct phenotypic changes in the CD3⁺CD8⁺ T cellsubset within the CTL stimulated with heteroclitic BCMA₇₂₋₈₀ (YLMFLLRKI(SEQ ID NO: 13)) peptide or heteroclitic TACI₁₅₄₋₁₆₂ (YLSADQVAL (SEQ IDNO: 16)) peptide in frequency of naïve (CD45RO⁻/CCR7⁺), central memory(CD45RO⁺/CCR7⁺), effector memory (CD45RO⁺/CCR7⁻) and terminal effector(CD45RO⁻/CCR7⁻) cells within the CD8⁺ T cell subsets in the CD3⁺ T cellcultures stimulated with the peptide. After 4 cycles of peptidestimulation, the frequency of effector memory CD3⁺CD8⁺ T cells wasincreased, associated with a corresponding decrease in naïve T cells(CD45RO⁻CCR7⁺/CD3⁺CD8⁺) and central memory T cells(CD45RO⁺CCR7⁺/CD3⁺CD8⁺). Thus, these results demonstrate that repeatedstimulation of CD3⁺ T cells with the selected heteroclitic BCMA or TACIpeptide results in distinct phenotypic changes and expansion of CD3⁺CD8⁺T cell subsets characteristic of antigen-specific CTL. (FIG. 12 and FIG.13 ).

Example 9. BCMA-Specific CTL and TACI-Specific CTL Induce CytotoxicActivity, Produce Th1-Type of Cytokines (IFN-γ, IL-2, TNF-α) andUpregulate 41BB Expression to MM Cells, in an HLA-A2-Restricted Manner

The peptide-specific CTL stimulated with heteroclitic BCMA₇₂₋₈₀(YLMFLLRKI (SEQ ID NO: 13)) peptide or heteroclitic TACI₁₅₄₋₁₆₂(YLSADQVAL (SEQ ID NO: 16)) were analyzed by flow cytometry for theirability to lyse myeloma cells and produce critical cytokines, which areinvolved in anti-tumor activities. The BCMA-CTL and TACI-specific CTLdemonstrated a significant increase in the frequency of cells expressingCD107a degranulation marker, a measure of cytotoxic activity, uponrecognition of HLA-A2⁺ U266 cells, which was higher than HLA-A2⁻ OPM2cells. An increased level of IFN-γ, IL-2, and TNF-α production wasdetected in BCMA-specific CTL and TACI-specific CTL to HLA-A2⁺ MM cells,but not to HLA-A2⁻ MM cells, demonstrating the immune responses are inan HLA-A2 restricted manner (FIG. 14 and FIG. 15 ).

Example 10. BCMA-Specific CTL Proliferate in Response to MM Cells inHLA-A2 Restricted and Antigen-Specific Manner

Functional activities of the peptide-specific CTL stimulated withheteroclitic BCMA₇₂₋₈₀ (YLMFLLRKI (SEQ ID NO: 13)) were further analyzedusing a CFSE-proliferation assay. The proliferation of CD8⁺ T cells inthe BCMA peptide-specific CTL was measured on day 4, evidenced by adecrease in fluorescence of the CF SE-labeled CTL (gated CFSE low)following stimulation with HLA-A2⁺ MM (U266), HLA-A2⁺ breast cancer(MDA-MB231) or HLA-A2⁻ MM (MM1S) cells. The BCMA-CTL induced asignificant CD8⁺ T cell proliferation in response to HLA-A2⁺ U266 MMcell line (proliferating cells: 46%). However, the CD8⁺ T cellsproliferation was not induced in response to MDA-MB231 or MM1S andstayed at a low level (11%-14%) as the cells cultured in media alone(10%). Taken together, these results suggest that the BCMA-CTL respondto myeloma cells specifically and their CD8⁺ T cells proliferation isHLA-A2-restricted and antigen-specific (FIG. 16 ).

Example 11. Higher Level of Cytotoxicity by BCMA-Specific CTL inCombination with Immune Agonist

The activity of peptide-specific CTL stimulated with heterocliticBCMA₇₂₋₈₀. (YLMFLLRKI (SEQ ID NO: 13)) was measured in treatment of thecells with anti-OX40 or anti-GITR for 48 hrs. The level of cytotoxicitywas measured by CD107a degranulation in the CD3⁺CD8⁺ T cells gated. Itwas observed that the CD107a degranulation was increased upon thetreatment of BCMA peptide-specific CTL with anti-OX40 (92%) or anti-GITR(55%) compared to untreated group (43%), suggesting that the combinationtreatment with immune agonists is helpful for inducing anti-tumoractivity of BCMA-CTL (FIG. 17 ).

Example 12. Selective Targeting of Multiple Myeloma by BCMA-SpecificCentral Memory CD8⁺ Cytotoxic T Lymphocytes

Despite recent advances in treatment of multiple myeloma (MM)incorporating novel therapies into the stem cell transplantationparadigm, ongoing DNA damage and genomic evolution underlie relapse inmany patients. Novel therapeutic approaches with distinct mechanisms ofaction are therefore needed. The constitutive or evolving geneticcomplexity, coupled with immune responsiveness of B cell malignancies,has stimulated the development of immunotherapeutic options in MMincluding monoclonal antibodies, bispecific antibodies, immunotoxins,and CAR T cells. Although MM patient-specific CAR T cell therapy hasachieved remarkable deep responses, durability of responses is notestablishes and they are labor-intensive, time-consuming, and expensive.To overcome these limitations, this example provides immunogenicpeptides-based cancer vaccines as an off-the-shelf immunotherapy fortreating patients more widely and efficiently. The peptide-basedtherapeutic approach does not have limitations of recombinant proteins,mRNA, or DNA-based vaccines, which require the processes ofinternalization, degradation of protein into optimal immunogenicpeptides to HLA, along with additional steps required for suitabletranslation (for mRNA) or transcription (for DNA). To overcome WICrestriction and treat a more diverse patient population using theimmunogenic epitope vaccine approach, peptide cocktails were pooled toinclude major HLA subtypes. Moreover, lenalidomide can augment peptidevaccine specific immune responses and memory cytotoxic T cell (CTL)activities, setting the stage for combination approaches with checkpointinhibitors and/or immune agonists. In addition, anti-tumor efficacytriggered by immunogenic peptides can be enhanced by their ability toinduce “epitope spreading” upon the generation of effector cells,whereby targeted lysed cancer cells release new antigenic epitopes whichare subsequently taken up, processed, and presented byantigen-presenting cells to a new repertoire of CTLs.

B cell maturation antigen (BCMA) is a member of the TNF receptorsuperfamily 17 (TNFRSF17) and is characterized as a type IIItrans-membrane protein containing cysteine-rich extracellular domainswith a central role in regulating B-cell maturation and differentiationinto plasma cells. As a receptor for the MM cell growth and survivalfactors B cell activating factor (BAFF) and a proliferation-inducingligand (APRIL), BCMA is required for the survival of MM cells, making ita promising therapeutic target. Nearly all MM tumor cells express BCMA,and it has been proposed as a marker for identification of tumor cells.Its selective expression on a subset of mature B and long lived plasmacells further suggest a favorable therapeutic index for BCMA directedtreatment approaches. At present BCMA is being targeted by severalimmunotherapeutic strategies including antibodies (naked antibodies,antibodies-drug conjugates, and bispecific antibodies) and cellulartherapies (chimeric antigen receptor T-cells), with promising clinicalresults even in relapsed refractory MM. In addition, serum soluble BCMAis elevated among patients with MM and chronic lymphocytic leukemia andcan serve as a prognostic marker and monitor of response. Finally, mostrecent studies indicate that BCMA is expressed in non-hemopoietictissue: BCMA is abnormally expressed in non-small cell lung cancer celllines and may play a role in the tumors through the ERK1/2 signalingpathway. These data support targeting BCMA in immunotherapeuticstrategies in MM and potentially BCMA expressing solid tumors as well.

This example provides a peptide-based immunotherapeutic approachtargeting BCMA by generating antigen-specific CD8⁺ CTL with effectiveand long-lasting immunity against MM cells. Novel immunogenic native andheteroclitic HLA-A2-specific BCMA peptides capable of elicitingMM-specific responses with highly effective anti-tumor activities wereidentified. Importantly, the heteroclitic BCMA₇₂₋₈₀ (ILMFLLRKI (SEQ IDNO: 13)) peptide demonstrated the highest level of immunogenicity, withthe greatest affinity/stability to HLA-A2 molecule and robust inductionof BCMA-specific memory CTL with poly-functional activities againstHLA-A2⁺ patients' MM cells and MM cell lines. The experiments show theframework for clinical application of this novel engineered immunogenicBCMA₇₂₋₈₀ peptide in cancer vaccine and adoptive immunotherapeuticprotocols, and provide long lasting memory anti-tumor immunity inpatients with MM or BCMA expressing cancers.

Particularly, this results show that tumor-associated antigens on CD138⁺tumor cells obtained from newly diagnosed MM patients (N=616) can beused to expand the breadth and extent of current multiple myeloma(MM)-specific immunotherapy. These experiments are designed to targetB-cell Maturation Antigen (BCMA), which promotes MM cell growth andsurvival, by generating BCMA-specific memory CD8⁺ CTL which mediateeffective and long-lasting immune response against MM cells. Here, theexperiment shows novel engineered peptides specific to BCMA, BCMA₇₂₋₈₀(YLMFLLRKI (SEQ ID NO: 13)) and BCMA₅₄₋₆₂ (YILWTCLGL (SEQ ID NO: 14))display improved affinity/stability to HLA-A2 compared to their nativepeptides and induce BCMA-specific CTL with increased activation (CD38,CD69) and co-stimulatory (CD40L, OX40, GITR) molecule expression.Importantly, the heteroclitic BCMA₇₂₋₈₀ specific CTL demonstratedpoly-functional Th1-specific immune activities [IFN-γ/IL-2/TNF-αproduction, proliferation, cytotoxicity] against MM, which were directlycorrelated with expansion of Tetramer⁺ and memory CD8⁺ CTL populations.When combined with anti-OX40 or anti-LAG3, the heteroclitic BCMA₇₂₋₈₀specific CTL displayed increased cytotoxicity against MM, especially bycentral memory CTL. These results provide the framework for clinicalapplication of heteroclitic BCMA₇₂₋₈₀ peptide, alone and in combinationwith anti-LAG3 and/or anti-OX40, in vaccination and adoptiveimmunotherapeutic strategies to generate long-lasting autologousanti-tumor immunity in patients with MM and other BCMA expressingtumors.

The following materials and methods were used in this example.

Materials and Methods Cell Lines

The MM cell lines, MM1S, OPM2, OPM1, H929, OCIMY5, RPMI, U266, KMS1,HSB2, McCAR and ANBL6, and a breast cancer cell line MDA-MB-231 wereobtained from ATCC (Manassas, Va.). The T2 cell line, a human B and Tcell hybrid expressing HLA-A2 molecules, was provided by Dr. J. Molldrem(University of Texas M. D. Anderson Cancer Center, Houston, Tex.). Thecell lines were cultured in DMEM (for MM and T2 cells; Gibco-LifeTechnologies, Rockville, Md.) or Leibovitz's L-15 (for MDA-MB231; ATCC,Manassas, Va.) media supplemented with 10% fetal calf serum (FCS;BioWhittaker, Walkersville, Md.), 100 IU/ml penicillin and 100 μg/mlstreptomycin (Gibco-Life Technologies).

Reagents

Fluorochrome conjugated anti-human BCMA, HLA-A2, CD3, CD8, CD38, CD40L,CD69, 41BB, CCR7, CD45RO, CD107a, IFN-γ, IL-2, TNF-α, PD1, LAG3, OX40and GITR monoclonal antibodies (mAbs) were purchased from BectonDickinson (BD) (San Diego, Calif.), LifeSpan Bioscience (Seattle, Wash.)or BioLegend (San Diego, Calif.). Live/Dead Aqua stain kit was purchasedfrom Molecular Probes (Grand Island, N.Y.). Recombinant human GM-CSF wasobtained from Immunex (Seattle, Wash.); and human IL-2, IL-4, IFN-α, andTNF-α were purchased from R&D Systems (Minneapolis, Minn.). BCMApeptide-specific Tetramer-PE was synthesized by MBL InternationalCorporation (Woburn, Mass.). Clinical grade mAb to LAG3 or OX40 wasprovided by Bristol-Myers Squibb (New York, N.Y.).

Synthetic Peptides

Native BCMA peptides [BCMA₆₄₋₇₂ (LIISLAVFV (SEQ ID NO: 1)), BCMA₆₉₋₇₇(AVFVLMFLL (SEQ ID NO: 2)), BCMA₉₋₁₇ (SQNEYFDSL (SEQ ID NO: 3)),BCMA₇₂-so (VLMFLLRKI (SEQ ID NO: 4)), BCMA₅₄₋₆₂ (AILWTCLGL (SEQ ID NO:5)), BCMA₁₁₄₋₁₂₀ (ILPRGLEYT (SEQ ID NO: 6))], heteroclitic BCMA peptides[hBCMA₇₂₋₈₀ (YLMFLLRKI (SEQ ID NO: 13)), hBCMA₅₄-62 (YILWTCLGL (SEQ IDNO: 14)), hBCMA₉-17 (YQNEYFDSL (SEQ ID NO: 22))] and HIV-Gage-85(SLYNTVATL (SEQ ID NO: 21)) were synthesized by standard fmoc(9-fluorenylmethyl-oxycarbonyl) chemistry, purified to >95% usingreverse-phase chromatography, and validated by mass-spectrometry formolecular weight (Biosynthesis, Lewisville, Tex.).

HLA A2 Affinity and Stability Assays

T2 cells were pulsed overnight with various doses of peptide plusβ2-microglobulin (3 μg/ml) (Sigma, St Louis, Mo.). Following overnightincubation, the cells were stained with HLA-A2-PE mAb and analyzed usinga FACSCanto™ flow cytometer (BD). Peptide/HLA-A2 complex stability wasmeasured on peptide loaded T2 cells at 0, 2, 4, 6 and 14 hourspost-brefeldin A treatment by staining with HLA-A2-PE mAb and flowcytometric analysis.

Generation of Dendritic Cells

Monocytes isolated from peripheral blood mononuclear cells (PBMC) werecultured for 7 days in the presence of 1,000 units/ml GM-CSF and 1,000units/ml IL-4 in RPMI-1640 medium (Gibco-Life Technologies) supplementedwith 10% FCS. Fresh media plus GM-CSF and IL-4 was added to the culturesevery other day. Mature DC (mDC) were obtained on day 7, following 3additional days incubation with 1,000 units/ml IFN-α plus 10 ng/mlTNF-α.

Induction of BCMA Peptide-Specific CTL

BCMA peptide-specific CTL (BCMA-CTL) were generated ex vivo by repeatedstimulation of CD3⁺ T cells obtained from HLA-A2⁺ donors withpeptide-pulsed antigen-presenting cells (APC). In brief, peptide (50μg/ml)-pulsed APC were irradiated (10 Gy) and used to stimulate T cellsat a 1 APC/peptide: 20 T cell ratio. The T cell cultures wererestimulated every 7 days and maintained in AIM-V medium supplementedwith 10% human AB serum (BioWhittaker) in the presence of IL-2 (50units/ml).

Phenotypic Analysis of BCMA Peptide-Specific CTL or Tumor Cells

Phenotypic characterization was performed on BCMA-CTL after stainingwith Live/Dead Aqua stain kit and fluorochrome conjugated anti-humanmAbs and Tetramer-PE. Alternatively, the MM and breast cancer cell lineswere stained with fluorochrome-conjugated BCMA or HLA-A2 mAb. Afterstaining, the cells were washed, fixed in 2% paraformaldehyde, andanalyzed by flow cytometry.

Cell Proliferation by Carboxy Fluorescein Succinimidyl Ester (CFSE)Tracking

BCMA-CTL were labeled with CFSE (Molecular Probes) and co-incubated withirradiated (10 Gy) tumor cells or peptide-pulsed APC in the presence ofIL-2 (10 units/ml). On day 4, 5, 6 or 8 of co-culture, cells wereharvested and stained with Live/Dead Aqua stain kit andCD3/CD8/CD45RO/CCR7 mAbs. The level of CD3⁺CD8⁺ CTL proliferation wasdetermined as a reduction in CFSE fluorescence intensity, as measured byflow cytometry.

CD107a Degranulation and Intracellular IFN-7/IL-2/TNF-α CytokinesProduction

The functional cytolytic activity of BCMA-CTL was measured by CD107adegranulation and Th1 cytokine production by flow cytometry. In brief,BCMA-CTL were co-incubated with tumor cells or T2/peptide in thepresence of CD107a mAb. After 1 hour incubation, CD28/CD49d mAb,brefeldin A, and Monensin (BD) were added for an additional 5 h. Cellswere harvested, washed in PBS, and incubated with mAbs specific to Tcell antigens. After surface staining, cells were fixed/permeabilized,stained with anti-IFN-γ/IL-2/TNF-α mAbs, washed with Perm/Wash solution(BD), fixed in 2% paraformaldehyde, and analyzed by flow cytometry.

Statistical Analysis

Results are presented as mean±SE. Groups were compared using unpairedStudent's t-test. Differences were considered significant when p<0.05.

BCMA Peptides Binding Affinity and Stability to HLA-A2 Molecules.

The full length BCMA protein sequence was evaluated to predict epitopeswith HLA-A2 affinity, extended half-time disassociation rates,proteasomal C terminal cleavage, and TAP transport using various searchsoftware programs including BIMAS and NetCTL. Among the six nativepeptides selected [BCMA₆₄₋₇₂ (LIISLAVFV), BCMA₆₉₋₇₇ (AVFVLMFLL),BCMA₉₋₁₇ (SQNEYFDSL), BCMA₇₂₋₈₀ (VLMFLLRKI), BCMA₅₄₋₆₂ (AILWTCLGL),BCMA₁₁₄₋₁₂₀ (ILPRGLEYT)], BCMA₇₂₋₈₀ (VLMFLLRKI) and BCMA₅₄₋₆₂(AILWTCLGL) showed the highest HLA-A2 binding affinity in adose-dependent manner. Among the heteroclitic peptides designed,heteroclitic BCMA₇₂₋₈₀. (YLMFLLRKI (SEQ ID NO: 13)) and heteroclitichBCMA₅₄-62 (YILWTCLGL (SEQ ID NO: 14)) displayed the highest increase inHLA-A2 binding affinity, as compared to their native peptides (n=3,p<0.05). In contrast, replacing the anchor motif in the non-HLA-A2specific BCMA₉₋₁₇ (SQNEYFDSL) to heteroclite BCMA₉₋₁₇ (YQNEYFDSL (SEQ IDNO: 22)) did not alter its HLA-A2 affinity status, indicating improvedHLA-A2 affinity by modification only within the HLA-A2-specificpeptides.

The HLA-A2 stability of BCMA₇₂₋₈₀ and BCMA₅₄₋₆₂ HLA-A2-specific peptidesafter brefeldin A treatment of the T2 cells pulsed with peptide wasassessed. Native BCMA₇₂₋₈₀ and BCMA₅₄₋₆₂ peptides displayed extendedHLA-A2 stability for greater than 6 hours, which was further enhanced byengineering into heteroclitic BCMA₇₂₋₈₀. (YLMFLLRKI (SEQ ID NO: 13)) andBCMA₅₄₋₆₂ (YILWTCLGL (SEQ ID NO: 14)). Overall, the highest level ofHLA-A2 affinity and stability was detected with the BCMA₇₂₋₈₀ (YLMFLLRKI(SEQ ID NO: 13)) at each time point tested, which was higher than theHLA-A2 positive control HIV-Gag-77-85 peptide.

BCMA-Specific CTL Generated with Heteroclitic BCMA₇₂₋₈₀ (YLMFLLRKI (SEQID NO: 13)) or BCMA₅₄₋₆₂ ILWTCLGL (SEQ ID NO: 14)) Show Increased T CellActivation and Costimulatory Molecule Expression.

Phenotypic characterization of heteroclitic BCMA₇₂₋₈₀ peptide-specificCTL (hBCMA₇₂₋₈₀ CTL) or heteroclitic hBCMA₅₄₋₆₂ peptide-specific CTL(hBCMA₅₄₋₆₂ CTL) was performed after the fourth round of peptidestimulation using flow cytometry. Both CTL populations displayedincreased activation marker (CD69, CD38) expression, with the highestupregulation detected on the hBCMA₇₂₋₈₀ CTL: CD38 increased to 80% frombaseline 23%; and CD69 increased to 38% from baseline 7% (FIG. 18A). Inaddition, the hBCMA₇₂₋₈₀ CTL showed higher expression of 41BB, CD40L,OX30, and GITR co-stimulatory molecules than hBCMA₅₄₋₆₂ CTL (FIGS. 18Band 18C).

In FIGS. 18A-18C, the CD3⁺ T cells obtained from HLA-A2⁺ individualswere stimulated weekly with irradiated APC pulsed with respectiveheteroclitic BCMA peptide, either BCMA₇₂₋₈₀ (YLMFLLRKI (SEQ ID NO: 13))or BCMA₅₄₋₆₂ (YILWTCLGL (SEQ ID NO: 14)). One week after the 4th cycleof stimulation, the CD3+CD8+ T cells were analyzed by flow cytometry.The expression of T cell activation markers (CD69, CD38) andcostimulatory molecules (41BB, CD40L, OX30, GITR) were evaluated on CD8+T cells. The results are demonstrated as a representative (FIGS. 18A and18B) or a summary of three independent experiments using BCMA-CTLgenerated from different individuals (N=3) (FIG. 18C).

Heteroclitic BCMA₇₂₋₈₀ Specific CTL Display Antigen-Specific Anti-TumorActivities in Response to MM Cell Lines.

The phenotype and activities of hBCMA₇₂₋₈₀ CTL were assessed after eachround of peptide stimulation. A gradual increase in the % CD3⁺CD8⁺ Tcells (FIGS. 25A-25B) and a corresponding decrease in % CD3⁺CD4⁺ T cells(FIGS. 26A-26B) was observed upon stimulation with heterocliticBCMA₇₂₋₈₀ (YLMFLLRKI (SEQ ID NO: 13)) in the specific CTL (n=3)generated. In parallel, phenotype analyses of target cells stained withBCMA mAb clones (ANC3B1, VICKY1, 19F2) showed high BCMA expression onH929, MMIS, U266 and OPM1 cell lines, but not on breast cancer cell line(MDA-MB231) (FIGS. 27A-27C). In evaluation of functional activities,hBCMA₇₂₋₈₀ CTL showed significantly (*p<0.05) higher CD3⁺CD8⁺ T cellsproliferation in response to HLA-A2⁺ BCMA⁺ U266 (49%) compared toHLA-A2⁺ BCMA⁺ MM1 S (7%), HLA-A2⁺ BCMA⁻ MDA-MB231 (9%), or media alone(6%) (FIGS. 19A-19D; Histogram). This HLA-A2-restricted and MM-specificCD8⁺ CTL proliferation was consistently observed in hBCMA₇₂₋₈₀ CTLgenerated from three HLA-A2⁺ individuals (FIG. 19E; Bar graphs). Inaddition, hBCMA₇₂₋₈₀ CTL demonstrated increases in CD8⁺ T cellsexpressing CD107a degranulation marker (47.1%) and producing Granzyme B(32.6%) and Perforin (29.9%) in response to HLA-A2⁺ U266, but not toHLA-A2⁺ MDA-MB231 cells (FIG. 19F). Consistent results in anti-tumoractivities were observed in hBCMA₇₂₋₈₀ CTL generated from other HLA-A2⁺individuals (N=5), as measured by IFN-γ/IL-2/TNF-α production, 41BBupregulation, and CD107a degranulation against BCMA⁺ MM cells in anHLA-A2 restricted manner. These data further demonstrate the inductionof MM-specific immune responses by heteroclitic BCMA₇₂₋₈₀ peptide.

In FIGS. 19A-9F, the BCMA-specific CTL generated by repeated stimulationwith heteroclitic BCMA₇₂₋₈₀ (YLMFLLRKI (SEQ ID NO: 13)) peptide wereexamined for their antigen-specific and HLA-A2-restricted CD8⁺ T cellsresponses by proliferation, CD107a degranulation, Granzyme B/perforinproduction, IFN-γ/IL-2/TNF-α production, and 41BB upregulation inresponse to BCMA⁺ MM cells or BCMA⁻ breast cancer cells. The results aredemonstrated as a representative (FIGS. 19A-19F) or a summary of threeindependent experiments using BCMA-CTL generated from differentindividuals (N=3).

Heteroclitic BCMA₇₂₋₈₀ CTL Functional Immune Responses Against HLA-A2⁺Patient MM Cells.

MM patients' CD138⁺ tumor cells were utilized as target cells toevaluate BCMA-specific CTL generated with respective heterocliticpeptides. Compared to heteroclitic BCMA₅₄₋₆₂, BCMA₇₂₋₈₀ peptide evokedmore robust antigen-specific CTL and anti-tumor activities againstpatient MM cells, as measured by CD107a degranulation (hBCMA₅₄₋₆₂ CTL13.8% vs. hBCMA₇₂₋₈₀ CTL 21.5%) and IL-2 production (4.4% vs. 16.3%,respectively) (FIG. 20A). The hBCMA₇₂₋₈₀ CTL consistently demonstratedhigher anti-MM activities against patient cells including CD107adegranulation, Granzyme B/IFN-γ/TNF-α upregulation (FIG. 20B), andperforin/IL-2 production (n=3) (FIGS. 20C-20H) in an HLA-A2 restrictedmanner. Thus, the anti-MM responses detected in the hBCMA₇₂₋₈₀ CTL wereconsistent with higher activation (CD69, CD38) and co-stimulatorymolecule expression (41BB, CD40L, OX40, GITR) (FIGS. 18A-18C). Thesedata provide additional evidence on the immunogenicity of heterocliticBCMA₇₂₋₈₀ and support its potential clinical application in novel MMtreatments.

In FIGS. 20A-20H, the heteroclitic BCMA peptide-specific CTL wereevaluated for their functional activities against patients' MM cells.The specific activities of BCMA-CTL were measured in response to CD138⁺tumor cells obtained from HLA-A2 negative or HLA-A2 positive MM patientsin relative to baseline response (stimulated with no tumor cells). Theresults are demonstrated as a representative (FIG. 20A, FIG. 20B) or asummary of three independent experiments using BCMA specific-CTLgenerated from different individuals (N=3) (FIGS. 20C-20H).

Heteroclitic BCMA₇₂₋₈₀ Specific CTL are Enriched for CD8⁺ Tetramer⁺ TCells with Robust Anti-MM Activities.

The hBCMA₇₂₋₈₀ CTL were further characterized for their phenotypes andanti-tumor activities within the Tetramer-positive population. TheTetramer-positive CTL displayed significantly increased the CD8⁺ T cellswith activation (CD38⁺: Tetramer⁺ vs. Tetramer⁻: 49.4% vs. 3.2%) andco-stimulatory molecule expression (CD40L⁺: 38.0% vs. 1.2%, 41BB: 24.7%vs. 1.9%, OX40: 46.2% vs. 1.7%, and GITR: 34.9% vs. 1.5%) (FIG. 21A).The hBCMA₇₂₋₈₀ CTL generated from several HLA-A2⁺ individuals (n=3)consistently demonstrated higher levels of anti-MM activities againstU266 MM cells by Tetramer-positive cells (83%, 97%, 97%; Donor A, B or CBCMA-CTL), as compared to Tetramer-negative cells (6%, 18%, 13%; DonorA, B or C BCMA-CTL) (FIG. 21B). The frequency of Tetramer-positive cellswithin either CD107a-positive or CD107a-negative CD8⁺ CTL was furtherevaluated. It was observed a significantly higher frequency of Tetramer+cells within the degranulating CD107a-positive CTL (82%, 98%, 98%; DonorA, B or C) compared to CD107a-negative CTL (1%, 2%, 1%; Donor A, B or CBCMA-CTL) (FIG. 21C). These results therefore confirm that the specificanti-MM activities of the hBCMA₇₂₋₈₀ CTL are contained within the BCMApeptide specific Tetramer-positive cells, which display upregulation ofCTL activation and co-stimulatory molecules.

In FIGS. 21A-21C, the heteroclitic BCMA₇₂₋₈₀ recognizingTetramer-positive CTL or non-recognizing Tetramer-negative CTL wereanalyzed for expression of CD38, CD40L, 41BB, OX40 and GITR on CD8⁺ Tcells (FIG. 21A). Anti-tumor activities of the heterocliticBCMA₇₂₋₈₀-specific CTL (N=3) were further characterized by measuringCD107 upregulation within Tetramer-positive CTL or Tetramer-negative CTLsubset (FIG. 21B); and by evaluating the status of Tetramer-positivitywithin CD107a-positive CTL or CD107a-negative CTL (FIG. 21C).

Heteroclitic BCMA₇₂₋₈₀ Peptide Induces MM-Specific Memory CD8⁺ CTL.

To characterize BCMA-specific CTL activities, experiments were performedto evaluate the composition of naïve: memory CTL subsets post-2 andpost-4 cycles of peptide stimulation, compared to baseline. A gradualprogressive phenotypic changes were detected within CD8⁺ T cells:progressing from naïve (CD45RO⁻CCR7⁺) at baseline [Donor 1—Naïve: 83.0%,CM: 0.4%, Donor 2—Naïve: 84.1%]; to central memory (CM; CD45RO⁺CCR7⁺)after 2 cycles of peptide stimulation [Donor 1—Naïve: 37.4%, CM: 32.1%,Donor 2—Naïve: 19.0%, CM: 49.6%]; and then to effector memory (EM;CD45RO⁺CCR7⁻) CTL after 4 cycles of stimulation (Donor 1—CM: 44.2%, EM:54.6%, Donor 2—CM: 18.3%, EM: 77.6%) (FIG. 22A). Overall, memory CD8⁺CTL development was gradually increased following each round (post-1, 2,3, 4 cycles) of heteroclitic BCMA₇₂₋₈₀ peptide stimulation (FIG.22B-22C), associated with a corresponding decrease in naïve T cells(FIG. 22D-22E). These results therefore demonstrate induction andgradual development of memory CTL upon the stimulation of T cells withheteroclitic BCMA₇₂₋₈₀ peptide.

In FIG. 22A-22E, the naïve: memory phenotype of heteroclitic BCMA₇₂₋₈₀CTL (Donor 1, Donor 2) were analyzed at baseline (no peptidestimulation) or one week after each cycle of peptide stimulation. Thepattern of phenotype changes, differentiation from naïve into memoryCD8⁺ T cells, and expansion of memory CTL were demonstrated in dot-plots(FIG. 22A) and bar graphs (FIGS. 22B-22E) after each cycle of BCMApeptide stimulation.

Central Memory hBCMA₇₂-so CTL Demonstrate the Greatest Anti-MMActivities

The specific memory T cell subsets within BCMA-specific CTL generatedfrom eight (N=8) different HLA-A2⁺ individuals were next characterizedfor their anti-MM activities. Compared to CD45RO⁻ non-memory CTL,CD45RO⁺ memory CTL demonstrated increased CD107a degranulation inresponse to HLA-A2⁺ U266 MM cells (non-memory vs. memory: 7.25% vs.28.2%) and HLA-A2⁺ McCAR MM cells (non-memory vs. memory: 4.14% vs.13.2%) (FIG. 23A; Donor A BCMA-CTL). The hBCMA₇₂₋₈₀ specific Tetramer+cells were mainly and consistently showed the CM phenotype in BCMA-CTLgenerated from different individuals (% CM within Tetramer+ cells—DonorB: 88.2%, Donor C: 97.4%, Donor D: 100%) (FIG. 23B). The CM CTL werealso evaluated for their functional activities against U266 MM cells.Importantly, the level of CD107a degranulation was directly associatedwith the frequency of CM cells (% CM within CD107a⁺ cells—Donor E:81.0%, Donor F: 82.6%, Donor G: 67.0%, Donor H: 41.5%) (FIG. 23C). Inaddition, the high responders (Donor E, Donor F) showing higher anti-MMactivities displayed increased frequency of BCMA peptide-specific CM CTLcompared to mid level responder (Donor G) or low level responder (DonorH). These results thus further indicate the distinct peptide-specificityand anti-MM activities induced by the CM subset generated by theheteroclitic BCMA₇₂₋₈₀ peptide.

In FIGS. 23A-23C, anti-MM activity of heteroclitic BCMA₇₂₋₈₀ CTL wasevaluated within the naïve: memory CD3⁺CD8⁺ T cell subsets in responseto HLA-A2⁺ MM cells (U266, McCAR; FIG. 23A). The frequency of centralmemory CD8⁺ T cells was analyzed in different CTL subsets ofheteroclitic BCMA₇₂₋₈₀ CTL (N=3); within Tetramer-positive orTetramer-negative CTL subsets (FIG. 23B) and within CD107a-positive orCD107a-negative CTL subsets (FIG. 23C).

Inhibition of LAG3 or Stimulation of OX40 Enhances Proliferation andAnti-MM Activities of hBCMA₇₂₋₈₀ CTL

Finally, experiments were performed to characterize the specific T cellsubset of BCMA-CTL which are highly responsive to MM cells. The CD8⁺ Tcell subset was gated, demonstrating HLA-A2-restricted MM specific CTLproliferation, and their Naïve: Memory subsets were characterized. Themost robust responding and highest proliferating hBCMA₇₂₋₈₀ CTL to U266MM cells were mainly within the CM subset (Donor 1: 97.4%, Donor 2:100%) (FIG. 24A), confirming the major role of CM subset within BCMAantigen-specific CTL in anti-MM activities. Next, experiments wereperformed to investiage the impact of a checkpoint inhibitor (anti-LAG3)or immune agonist (anti-OX40) on these memory T cells. The hBCMA₇₂₋₈₀CTL treated with either anti-LAG3 or anti-OX40 demonstrated enhancedcytotoxic activity, especially by memory CTL against HLA-A2⁺ U266 MMcells (Untreated 28.2% vs. anti-LAG3 treated 35.8% vs. anti-OX40 treated39.5%), and against HLA-A2⁺ McCAR MM cells (Untreated 13.2% vs.anti-LAG3 treated 14.5% vs. anti-OX40 treated 20.0%) (FIG. 24B).Interestingly, the checkpoint inhibitor and immune agonist did notinduce enhance the anti-MM responses of non-memory cells withinBCMA-CTL. Lastly, the beneficial effect of anti-LAG3 and anti-OX40 wasfurther investigated within CM and EM subsets of hBCMA₇₂₋₈₀ CTL. Eithertreatment induced greater impact on BCMA-specific CM cells compared toEM cells, evidenced by higher CD107a degranulation in response toanti-LAG3 or anti-OX40 treatment (FIG. 24C). These results thereforesupport the utility of anti-LAG3 or anti-OX40 antibody in combinationwith hBCMA₇₂₋₈₀ peptide induced CTL to further enhance anti-MMactivities within the BCMA-specific CM subset.

In FIGS. 24A-24C, the specific subset inducing MM-specific CD8⁺ T cellproliferation was identified within heteroclitic BCMA₇₂₋₈₀ specific CTLin response to U266 cells (FIG. 24A). Furthermore, the heterocliticBCMA₇₂₋₈₀ CTL was evaluated in combination with anti-LAG3 or anti-OX40for their modification of anti-myeloma activities by memory T cells(FIG. 24B) or central memory T cell subset (FIG. 24C).

Discussion

Even in patients with refractory MM relapsing after allotransplantation,long-lasting responses have been achieved with the infusion of donorlymphocytes (DLI). These early encouraging results of DLI have providedthe framework for evaluation of active-specific immunotherapy approachesto treat MM. Cancer targeting vaccines, one such active-specificimmunotherapy approach, have demonstrated the ability to induce highlyeffective CD8⁺ CTL with anti-tumor activities. The success ofvaccination depends on selection of the appropriate patient population,targeting antigens expressed selectively on tumor, and utilizingcombination approaches to effectively induce and maintainantigen-specific memory anti-tumor immune responses. This disclosureprovides immunogenic HLA-A2 and HLA-A24 specific peptides derived fromXBP1, CD138 and CS1 antigens, which are highly over-expressed in MM andsolid tumors including breast, pancreatic, and colon cancers, anddemonstrated their ability to induce the peptides-specific CD8⁺ CTL withanti-tumor activities against HLA-A2⁺ or HLA-A24⁺ tumor cells both inpreclinical and clinical studies. In addition, combination studies ofpeptide stimulation/vaccination with immune modulatory drugs such aslenalidomide or with histone deacetylase 6 inhibitor ACY241 enhanced thepeptides-specific CTL activities against tumor cells. The experimentsdemonstrated that combinations of peptide stimulation with eitherLenalidomide or ACY241 augmented antigens-specific CD8⁺ T cell activityassociated with upregulation of transcriptional regulators such asT-bet/Eomes and with activation of AKT, which links antigen-specific CTLdifferentiation to FOXO, mTOR and Wnt/β-catenin signaling pathways.Importantly, these effects were confined primarily to antigen-specificCD45RO⁺ memory CTL, with the most robust increases in IFN-γ and granzymeB production and CD8⁺ T cell proliferation in response to tumor cellsobserved mainly within the specific CM subset.

Due to the encouraging preclinical results, the XBP1/CD138/CS1multipeptide vaccine has been evaluated, alone and in combination withlenalidomide, in clinical trials to treat patients with smoldering MM(SMM), as well as in combination with anti-PD1 in clinical trials totreat patients with triple negative breast cancer. In patients with SMM,the multipeptide vaccine was well tolerated and immunogenic as amonotherapy, evidenced by enhanced frequency of Tetramer⁺ CD8⁺ CTL withIFN-γ production; moreover, combination with lenalidomide triggeredhigher mean fold increases in CD8⁺ T cells with tetramer-positivity andIFN-γ production. Importantly, CD45RO⁺ memory CTL specific to theXBP1/CD138/CS1 peptides were induced by the peptide vaccine, and furtherenhanced in combination with lenalidomide. Although stable disease andresponses have been observed in SMM, randomized trials are needed toassess whether time to progression from SMM to active disease can beprolonged by the peptide vaccination.

To expand the MM-specific immunotherapy beyond XBP1/CD138/CS1 antigens,the disclosure also identified additional tumor associated antigens onCD138⁺ tumor cells from newly diagnosed MM patients (N=616). Here thedisclosure provides the identification and characterization of animmunotherapeutic strategy targeting BCMA, selectively expressed onnormal and malignant plasma cells and the target of several currentimmune treatments in MM. The examples provide highly immunogenicengineered BCMA-specific nanopeptides, heteroclitic BCMA₇₂₋₈₀ (YLMFLLRKI(SEQ ID NO: 13)) and BCMA₅₄₋₆₂ (YILWTCLGL (SEQ ID NO: 14)) with highlyimproved HLA-A2 affinity/stability from their native BCMA peptides.These peptides evoke BCMA-specific CTL, increased BCMA-specificTetramer⁺ cells, enhanced CD107 degranulation, Th1-type cytokines(IFN-γ/IL-2/TNF-α) production, and proliferation to MM cells in anHLA-A2-restricted manner. Most importantly, the increase ofBCMA-specific memory CD8⁺ CTL, both CM and EM cells, along with thecapacity of self-renewal and response to MM cells, strongly support thepotential of heteroclitic BCMA peptide in novel vaccination and/orimmunotherapeutic approaches in MM. Indeed, the disclosure providesclinical protocols with heteroclitic BCMA₇₂₋₈₀ peptide vaccination,harvest and expansion of BCMA-specific CM cells ex vivo, reinfusion ofthese CM cells as adoptive immunotherapy, and then vaccination with theBCMA peptide as needed thereafter to assure their persistence toeffectively treat MM patients.

It has been observed that BCMA-specific memory CD8⁺ CTL expressed keymolecules modulating T cells function, both for co-stimulation andimmune suppression. The highest induction of co-stimulatory and immunecheckpoint molecules was detected on CM subset within hBCMA₇₂₋₈₀peptide-specific CTL, which is the population demonstrated highlyeffective poly-functional activities against MM. Importantly, thesefindings indicated the potential of combination therapy of BCMA-CTL withcheckpoint inhibitors or immune agonists to enhance their functionalanti-MM activities. This may be particularly relevant, given the recentconcerns when combining PD-1 checkpoint inhibitor with immunomodulatorydrugs lenalidomide or pomalidomide or with Ab daratumumab, wheretoxicities have curtailed studies. Here, the examples attempted totargeting alternative inhibitory receptors and suppressive mechanismswithin the MM tumor microenvironment. In particular, LAG3 (CD223) is thethird inhibitory receptor to be targeted in the clinic, following CTLAand PD1/PD-L1 and was expressed on BCMA-specific CM CTLs. In parallel,immune agonists, especially the co-stimulatory tumor necrosis factorreceptors targeting OX40 (CD134), 41BB (CD137) and GITR (CD357), havereceived considerable attention for their therapeutic utility inenhancing anti-tumor immune responses; among these, anti-OX40 mAb hasrecently demonstrated encouraging efficacy in induction of tumorregression by boosting effector T cell expansion and functionalanti-tumor activities in several pre-clinical studies. Importantly, aclinical grade anti-LAG3 and anti-OX40 (provided by Bristol-MyersSquibb; New York, N.Y.) was used to evaluate functional activities ofheteroclitic BCMA₇₂₋₈₀ specific CTL to MM cells. The ex vivo experimentsdemonstrated that both anti-LAG3 and anti-OX40 increased functionalactivity specifically of memory CTL within the BCMA-CTL against MMcells, without affecting the activity of non-memory CTL. The impact onBCMA-CTL generated from multiple HLA-A2⁺ individuals' T cells wasgreater after treatment with anti-OX40 than anti-LAG3, and greater on CMversus EM subset within BCMA specific CTL. These studies provide theframework for scientifically-informed combination clinical trials ofBCMA peptide specific immunotherapy with the immune agonist orcheckpoint inhibitor.

In summary, these experiments identified and validated novel immunogenicHLA-A2-specific engineered BCMA peptides, which are capable of inducingantigen-specific CD8⁺ CTL with functional anti-tumor activities againstMM cells. These results provide the framework for therapeuticapplication of these highly immunogenic heteroclitic BCMA peptides in MMpatients as vaccines and/or as stimuli for expansion of autologousantigen-specific memory CTL. They further support the potential utilityof combinations incorporating BCMA peptide vaccine or BCMA-specificadoptive T cells immunotherapy with anti-OX40 and/or anti-LAG3 toenhance BCMA directed anti-MM responses.

Example 13. HLA-A2-Specific Immunogenic TACI Peptide for ElicitingTACI-Specific CD8⁺ Cytotoxic T Lymphocytes

Experiments were performed to demonstrate that novel immunogenicengineered heteroclictic TACI₁₅₄₋₁₆₂ (YLSADQVAL (SEQ ID NO: 16)) peptidecan induce antigen-specific memory CD8⁺ CTL with robust poly-functionalimmune responses against MM. These results in this example provide theframework for therapeutic application of heteroclitic TACI peptides inMM patients and support the therapeutic application of TACIpeptides-specific vaccine or TACI peptides-specific adoptive T cellsimmunotherapy to treat the patients with myeloma or other diseasesexpressing TACI.

The following materials and methods were used in the examples.

Materials and Methods Cell Lines

The HLA-A2⁺ (U266 and McCAR) and HLA-A2⁻ (OPM2 and RPMI) MM cell lineswere obtained from ATCC (Manassas, Va.). The T2 cell line, a human B andT cell hybrid expressing HLA-A2, was provided by Dr. J. Molldrem(University of Texas M. D. Anderson Cancer Center, Houston, Tex.). Thecell lines were cultured in DMEM (for MM and T2 cells; Gibco-LifeTechnologies, Rockville, Md.) media supplemented with 10% fetal calfserum (FCS; BioWhittaker, Walkersville, Md.), 100 IU/ml penicillin and100 μg/ml streptomycin (Gibco-Life Technologies).

Reagents

Fluorochrome conjugated anti-human monoclonal antibodies (mAbs) werepurchased from Becton Dickinson (BD) (San Diego, Calif.), LifeSpanBioscience (Seattle, Wash.) or BioLegend (San Diego, Calif.). Live/DeadAqua stain kit was purchased from Molecular Probes (Grand Island, N.Y.).Recombinant human GM-CSF was obtained from Immunex (Seattle, Wash.) andhuman IL-2, IL-4, IFN-α and TNF-α were purchased from R&D Systems(Minneapolis, Minn.). TACI peptide-specific Tetramer-PE was synthesizedby MBL International Corporation (Woburn, Mass.).

Synthetic Peptides

Native TACI peptides [TACI₁₇₈₋₁₈₆ (FLVAVACFL (SEQ ID NO: 7)),TACI₁₇₄₋₁₈₂ (VLCCFLVAV (SEQ ID NO: 8)), TACI₁₅₄₋₁₆₂ (KLSADQVAL (SEQ IDNO: 9)), TACI₁₆₆₋₁₇₄ (TLGLCLCAV (SEQ ID NO: 10)), TACI₁₆₁₋₁₆₉ (ALVYSTLGL(SEQ ID NO: 11)), TACI₁₅₅₋₁₆₃ (LSADQVALV (SEQ ID NO: 12))], heterocliticTACI peptides [TACI₁₇₈₋₁₈₆ (YLVAVACFL (SEQ ID NO: 15)), TACI₁₅₄₋₁₆₂(YLSADQVAL (SEQ ID NO: 16)), TACI₁₆₆₋₁₇₄ (YLGLCLCAV (SEQ ID NO: 17))]and HIV-Gage-85 (SLYNTVATL (SEQ ID NO: 21)) peptides (HLA-A2-specificpositive control peptide) were synthesized by standard fmoc(9-fluorenylmethyl-oxycarbonyl) chemistry, purified to >95% usingreverse-phase chromatography and validated by mass-spectrometry formolecular weight (Biosynthesis, Lewisville, Tex.).

HLA A2 Affinity and Stability Assays

T2 cells were pulsed overnight with various concentrations of peptideplus β2-microglobulin (3 μg/ml) (Sigma, St Louis, Mo.). Followingovernight incubation, the cells were stained with HLA-A2-PE mAb andanalyzed using a FACSCanto™ flow cytometer (BD). The stability ofpeptide/HLA-A2 complex binding was measured on peptide loaded T2 cellsat 0, 2, 4, 6 and 14 hours post-brefeldin A treatment followed bystaining with HLA-A2-PE mAb and flow cytometric analysis.

Generation of Dendritic Cells

Monocytes isolated from peripheral blood mononuclear cells (PBMC) werecultured for 7 days in the presence of 1,000 units/ml GM-CSF and 1,000units/ml IL-4 in RPMI-1640 medium (Gibco-Life Technologies) supplementedwith 10% FCS. Fresh media plus GM-CSF and IL-4 was added to the culturesevery other day. Mature DC (mDC) were obtained on day 7, following 3additional days incubation with 1,000 units/ml IFN-α plus 10 ng/mlTNF-α.

Induction of TACI Peptide-Specific CTL

TACI peptide-specific CTL (TACI-CTL) were generated ex vivo by repeatedstimulation of enriched CD3⁺ T cells obtained from HLA-A2⁺ donors withpeptide-pulsed antigen-presenting cells (APC). In brief, peptide (50μg/ml)-pulsed APC were irradiated (10 Gy) and used to stimulate T cellsat a 1:20 APC/peptide-to-T cell ratio. The T cell cultures wererestimulated every 7 days and maintained in AIM-V medium supplementedwith 10% human AB serum (BioWhittaker) in the presence of IL-2 (50units/ml).

Phenotypic Analysis of TACI Peptide-Specific CTL or Stimulatory TumorCells

Phenotypic characterization was performed on the MM target cells toconform TACI expression. Phenotypic characterization was performed onthe TACI-CTL after staining with Live/Dead Aqua stain kit andfluorochrome conjugated anti-human mAbs. After staining, the cells werewashed, fixed in 2% paraformaldehyde, and analyzed by flow cytometry.

Cell Proliferation by Carboxy Fluorescein Succinimidyl Ester (CFSE)Tracking

TACI-CTL were labeled with CFSE (Molecular Probes) and co-incubated withirradiated (10 Gy) MM cells or peptide-pulsed APC in the presence ofIL-2 (10 units/ml). On day 4, 5, 6 or 8 of co-culture, the cells wereharvested and stained with Live/Dead Aqua stain kit and fluorochromeconjugated anti-human mAb specific to CD3, CD8, CD45RO and CCR7. Thelevel of CD3⁺CD8⁺ CTL proliferation was determined as a reduction inCFSE fluorescence intensity, as measured by flow cytometry.

CD107a Degranulation and Intracellular IFN-7/IL-2/TNF-α CytokinesProduction

The functional cytolytic activity of TACI-CTL was measured by CD107adegranulation (cytotoxicity) and production of Th1 cytokines by flowcytometry. In brief, TACI-CTL were co-incubated with tumor cells orT2/peptide in the presence of CD107a mAb. After 1 hour, CD28/CD49d mAb,brefeldin A and Monensin (BD) were added for an additional 5 hoursincubation. Cells were harvested, washed in PBS, and incubated withfluorochrome conjugated mAbs to key T cell markers. After surfacestaining, cells were fixed/permeabilized, stained withanti-IFN-γ/IL-2/TNF-α mAbs, washed with Perm/Wash solution (BD), fixedin 2% paraformaldehyde, and analyzed by flow cytometry.

Statistical Analysis

Results are presented as mean±SE. Groups were compared using unpairedStudent's t-test. Differences were considered significant when p<0.05.

Results

Identification of Heteroclitic TACI₁₅₄₋₁₆₂ Peptide with the HighestBinding Affinity and Stability to HLA A2 Molecules

The full length TACI protein sequence was evaluated to predict epitopeswith HLA-A2 affinity, extended half-time disassociation rates,proteasomal C terminal cleavage and TAP transport using various searchsoftware programs including BIMAS and NetCTL. Among the six nativepeptides selected [TACI₁₇₈₋₁₈₆ (FLVAVACFL (SEQ ID NO: 7)), TACI₁₇₄₋₁₈₂(VLCCFLVAV (SEQ ID NO: 8)), TACI₁₅₄₋₁₆₂ (KLSADQVAL (SEQ ID NO: 9)),TACI₁₆₆₋₁₇₄ (TLGLCLCAV (SEQ ID NO: 10)), TACI₁₆₁₋₁₆₉ (ALVYSTLGL (SEQ IDNO: 11)), TACI₁₅₅₋₁₆₃ (LSADQVALV (SEQ ID NO: 12))], all peptidesexcepting for TACI₁₆₁₋₁₆₉ displayed the HLA-A2 specificity, however thehighest level of affinity was detected by TACI₁₆₆₋₁₇₄. In order toimprove the binding and stability of the peptide to HLA-A2 molecules,three heteroclitic peptides were designed including TACI₁₇₈₋₁₈₆(YLVAVACFL (SEQ ID NO: 15)), TACI₁₅₄₋₁₆₂ (YLSADQVAL (SEQ ID NO: 16)) andTACI₁₆₆₋₁₇₄ (YLGLCLCAV (SEQ ID NO: 17)) peptides, synthesized andevaluated their HLA-A2 affinity. The heteroclitic TACI₁₅₄₋₁₆₂ peptidedisplayed greatly enhanced affinity from its native form at the peptidedoses of 50 μg/ml and 100 μg/ml. The HLA-A2 affinity of heterocliticTACI₁₅₄₋₁₆₂ peptide was similar to the affinity of the HIV-Gag₇₇₋₈₅(SLYNTVATL (SEQ ID NO: 21)), which was served as HLA-A2-specificpositive control peptide. However, the engineered heterocliticTACI₁₇₈₋₁₈₆ and TACI₁₆₆₋₁₇₄ peptides showed no significant improvementin HLA-A2 affinity as compared to their native peptides. The selectedTACI peptides (50 μg/ml) were further evaluated for their affinity andstability to HLA-A2 molecule. The highest HLA-A2 binding affinity(time=0) and stability (2, 4, 6 and 18 hours) was seen with theheteroclitic TACI₁₅₄₋₁₆₂ as compared to its native peptide. Overall, theHLA-A2 affinity stability of heteroclitic TACI₁₅₄₋₁₆₂ was similar toHLA-A2 positive control HIV-Gag₇₇₋₈₅ peptide at the various time points.Based on the highest level of HLA-A2 affinity and stability among theTACI peptides investigated, the heteroclitic TACI₁₅₄₋₁₆₂ was selectedfor further evaluation of its immunogenic potential to induce theantigen-specific effector T cells against MM.

Anti-Tumor Activities of Heteroclitic TACI₁₅₄₋₁₆₂ Peptide-Specific CTLThrough Development and Expansion of Antigen-Specific Memory CD8⁺ CellsDifferentiated from Naïve CD8⁺ Cells.

Phenotypic changes in CD8⁺ T cell were evaluated within Naïve: Memorysubsets, following weekly stimulation of enriched CD3⁺ T cells fromHLA-A2⁺ donors with the heteroclitic TACI₁₅₄₋₁₆₂ (YLSADQVAL (SEQ ID NO:16)). At baseline [prior to peptide stimulation], the majority (84.1%)of the CD8⁺ T cells were found as Naïve (CD45RO⁻CCR7⁺) cell subset. Uponthe stimulation with heteroclitic TACI₁₅₄₋₁₆₂ peptide, it was observedthat a gradual differentiation of Naïve CTL into central memory CTL (CM;CD45RO⁺CCR7⁺CD3⁺CD8⁺) and then effector memory CTL (EM;CD45RO⁺CCR7⁻/CD3⁺CD8⁺) within heteroclitic TACI₁₅₄₋₁₆₂ peptide-specificCTL (hTACI₁₅₄₋₁₆₂ CTL). Development of memory CTL was detected after 3cycles of peptide stimulation [Naïve: 2.2%, CM: 47.8%, EM: 52.4%] withthe heteroclitic TACI₁₅₄₋₁₆₂ peptide. Further differentiation wasobserved from naïve CTL into the memory CTL subsets after 4 cycles ofpeptide stimulation [Naïve: 0.72%, CM: 24.4%, EM: 74.1%] and 5 cycles ofstimulation [Naïve: 0.57%, CM: 7.36%, EM: 69.7%] (FIG. 29A), supportingthe potential of peptide to develop memory T cells. The immunogenicityof heteroclitic TACI₁₅₄₋₁₆₂ peptide was further evaluated in T cellsobtained from additional HLA-A2⁺ donors (N=3) (FIG. 29B). The gradualdecrease in naïve CD8⁺ CTL and increase in memory CD8⁺ CTL (both centralmemory and effector memory CTL) were verified in all the individuals' Tcells tested, following stimulation with the heteroclitic TACI₁₅₄₋₁₆₂peptide. Thus, these results confirm the capacity of heterocliticTACI₁₅₄₋₁₆₂ peptide to develop memory CD8⁺ T cells.

Next, experiments were performed to evaluate the immune functionalcapacity of the heteroclitic TACI₁₅₄₋₁₆₂-specific CTL in response tomyeloma cells. The proliferation of CD8⁺ CTL was detected in theheteroclitic TACI₁₅₄₋₁₆₂ peptide-specific CTL, especially in memory CTLincluding central memory and effector memory CTL subsets, upon theco-culture with HLA-A2⁺ U266 MM cells. The proliferative capacity of theTACI-CTL continued to increase, following more cycles of TACI₁₅₄₋₁₆₂peptide stimulation [2′ stimulation: Proliferating CM—38.1%,Proliferating EM—19.2%; 3rd stimulation: Proliferating CM—54.4%,Proliferating EM—85.2%; 4th stimulation: Proliferating CM—64.4%,Proliferating EM—86.9%] (FIG. 29C). Thus, these results further supportthe immune function of heteroclitic TACI₁₅₄₋₁₆₂ peptide-specific CTLthrough development and continuous expansion of memory CD8⁺ T cells inresponse to myeloma cells.

Proliferation of TACI-Specific Tetramer⁺ CTL in Heteroclitic TACI₁₅₄₋₁₆₂Specific CTL Demonstrating Polyfunctional and Th1-Specific Anti-MyelomaActivities in HLA-A2-Restricted Manner.

The functional activities of hTACI₁₅₄₋₁₆₂ CTL were examined for theirpolyfunctional immune responses against myeloma cells. The hTACI₁₅₄₋₁₆₂CTL demonstrated HLA-A2 restricted degranulation (CD107a upregulation)against HLA-A2⁺ McCAR MM cells, which is directly associated withcytotoxic activity against tumor cells, as compared to HLA-A2⁻ RPMI(8.24%) or media alone (5.03%) (FIG. 30A). In addition, the hTACI₁₅₄₋₁₆₂CTL demonstrated a higher production of Th1-type of cytokines, IFN-γ(15.0%), TNF-α (17.5%) and IL-2 (14.9%), in response to HLA-A2⁺ McCAR,as compared to HLA-A2⁻ RPMI (IFN-γ 8.26%, TNF-α 7.80%, IL-2 6.42%) ormedia alone (IFN-γ 5.76%, TNF-α 5.52%, IL-2 6.24%). The specific anti-MMactivities, as measured by CD107a degranulation and IFN-γ production,were consistently observed in hTACI₁₅₄₋₁₆₂ CTL generated from differentHLA-A2⁺ individuals (N=5), against HLA-A2⁺ MM cells (McCAR) as comparedto the MHC mis-matched HLA-A2⁻ MM cells (RPMI) (FIG. 30B, 3C). Toconfirm the HLA-A2-specific anti-MM activities of heteroclitichTACI₁₅₄₋₁₆₂ CTL, experiments were performed to evaluate their immunefunctional activities against additional myeloma cell lines includingHLA-A2⁺ U266 and HLA-2⁻ OPM2 cells. The same pattern ofHLA-A2-restricted functional anti-tumor activities were observed againstthe myeloma cells in hTACI₁₅₄₋₁₆₂-CTL generated from HLA-A2⁺ Donor 1(FIG. 31A) as well as a total of five different HLA-A2⁺ individuals(FIGS. 31B-31E). Furthermore, the hTACI₁₅₄₋₁₆₂ CTL stained positive forTACI₁₅₄₋₁₆₂ peptide-specific Tetramer⁺ CTL and these Tetramer⁺ cellsdemonstrated a high level of CD107a degranulation and proliferation inresponse to HLA-A2⁺ U266 myeloma cells (FIG. 31F). Thus, these resultsdemonstrate the HLA-A2-restricted immunogenicity of heterocliticTACI₁₅₄₋₁₆₂ peptide to evoke antigen-specific CTL with poly-functionalactivities (cytotoxicity, Th-1 type cytokine production) against MMcells, which supporting the potential therapeutic application of theimmunogenic heteroclictic TACI₁₅₄₋₁₆₂ (YLSADQVAL (SEQ ID NO: 16))peptide in myeloma patients.

Example 14. BCMA Heteroclitic Peptide Encapsulated Nanoparticle EnhancesAntigen Stimulatory Capacity and Tumor-Specific CD8⁺ Cytotoxic TLymphocytes Against Multiple Myeloma

B-cell Maturation Antigen (BCMA), a member of the tumor necrosis factor(TNF) receptor superfamily and the receptor for binding of B cellactivating factor (BAFF) and the proliferation-inducing ligand (APRIL),is a promising therapeutic target for MM. BCMA has restricted expressionpattern on MM cells and plasma cells and has a role in promoting MMcells growth, survival, and drug resistance.

The present disclosure has identified nanomedicine-based therapeuticstargeting BCMA as a promising area of translational research toeffectively evoke and augment anti-tumor responses in MM patients.Several nanomedicines are available and more advanced nanoparticleconstructs are under development for antigen encapsulation. To this end,this example provides novel engineered peptides specific to BCMA, andused a heteroclitic BCMA₇₂₋₈₀ (YLMFLLRKI (SEQ ID NO: 13)) peptideencapsulated nanoparticle-based cancer vaccine to overcome thelimitations of free peptide vaccines including poor peptide stability,susceptibility to enzyme degradation, and low antigen uptake anddelivery. Furthermore, the nanotechnology-based cancer vaccine wasdeveloped to induce more robust BCMA-specific CD8⁺ cytotoxic Tlymphocytes (CTL) activities in MM patients, with more sustained antigenrelease and increased bioavailability and presentation of theimmunogenic peptide. Here, experiments are performed to examine thepotential of a novel nanomedicine-based therapeutic delivery systemspecific to BCMA antigen to treat patients with MM. The purpose of thisexample was to design the optimal nanoparticle encapsulated BCMA antigenconstructs to efficiently evoke BCMA-specific CD8⁺ CTL with functionalanti-myeloma activities.

The results show that nanoparticles [liposome orpoly(D,L-lactide-co-glycolide) (PLGA)] with different antigen-releasekinetics demonstrated their capacity to effectively deliver heterocliticBCMA peptide to antigen-presenting cells and evoke BCMA antigen-specificCTL with anti-MM activities. The heteroclitic BCMA peptide encapsulatednanoparticles demonstrated a higher uptake by human dendritic cells thanfree peptide, with the highest uptake mediated with liposome-basednanoparticles. In contrast, BCMA-specific CTL induced with PLGA-basednanoparticle demonstrated the highest functional activities and specificimmune responses against MM cells. Importantly, the PLGA/BCMA peptidenanoparticle-induced BCMA-specific CTL displayed greater CD107adegranulation, antigen-specific CD8⁺ CTL proliferation, and Th-1 typecytokines (IFN-γ, IL-2, TNF-α) production in response to MM patients'tumor cells and MM cell lines than BCMA-CTL generated with free BCMApeptide or liposome/BCMA peptide nanoparticle. CD28 costimulatorymolecules upregulation, Tetramer⁺ CTL generation, and peptide-specificresponses within the BCMA-CTL generated by PLGA/BCMA nanoparticles werealso greater than BCMA-CTL generated with free BCMA peptide orliposome/BCMA peptide nanoparticle. Furthermore, the PLGA/BCMAnanoparticles triggered a more robust induction of antigen-specificmemory CD8⁺ T cells, which demonstrated significantly higher anti-tumoractivities, evidenced by CD107a degranulation and IFN-γ production thannon-memory CD8⁺ T cells within the BCMA-CTL. The induction of centralmemory CTL with anti-tumor activities by PLGA/BCMA peptide wereassociated with the optimal peptide release kinetics and enhancedimmunogenicity.

These results therefore demonstrate that the heteroclitic BCMA peptideencapsulated nanoparticle strategy enhances peptide delivery intodendritic cells and subsequently to T cells, thereby inducingBCMA-specific central memory CTL with poly-functional activities againstMM.

These results also demonstrate the utility of nanotechnology usingencapsulated heteroclitic BCMA peptide to enhance the immunogenicity ofBCMA peptide-specific therapeutics against MM. Importantly, theobservations provide the framework for therapeutic application of PLGAnanoparticle-based heteroclitic BCMA peptide delivery to enhance theBCMA-specific memory T cell immune responses, overcome the limitationsof current peptide-based cancer vaccine and adoptive immunotherapy, andimprove patient outcome in MM.

The following methods and materials were used in this example.

Materials and Methods Materials

Poly(D,L-lactide-co-glycolide) (PLGA, molecular weight 23,000, copolymerratio 50:50) was purchased from Birmingham Polymers (Birmingham, Ala.).Polyvinyl alcohol (PVA, average molecular weight 30,000-70,000),trifluoroacetic acid, acetonitrile, lipopolysaccharide, and L-15(Leibovitz) medium were purchased from Sigma-Aldrich (St Louis, Mo.).

Formulation and Characterization of BCMA Peptide-Loaded PLGA-NP.

Immunogenic heteroclitic hBCMA₇₂₋₈₀ (YLMFLLRKI (SEQ ID NO: 13)) peptidewas used to produce BCMA antigen-specific nanoparticle preparations forgeneration of BCMA-specific cytotoxic T lymphocytes (CTL). A doubleemulsion-solvent technique was used to formulate hBCMA₇₂₋₈₀ peptide withPLGA-NP, along with Poly(vinyl alcohol) (PVA) to stabilize the emulsion.Heteroclitic hBCMA₇₂₋₈₀ peptide with PLGA-NPs was formulatedsubstantially as previously described, (Sahoo S K, et al. 2004). Inbrief, PLGA (50:50 lactide-to-glycolide ratio) and peptide or blank PLGAitself were emulsified in Dichloromethane (DCM), and the mixture wasresuspended in 2% PVA to form an oil and water emulsion. Theemulsification process was completed using a micro-tip probeultra-sonicator at 55 watts for 10 minutes in an ice bucket. Theemulsion was stirred for 3 hr at room temperature to allow evaporationof DCM and formation of PLGA-NPs. The peptide-loaded PLGA-NPs wererecovered by ultracentrifugation at 30,000 rpm for 30 min at 4° C. todeplete PVA and free peptide, washed, and resuspended in PBS. Toevaluate PLGA-NP structure, the nanoparticle formulations werelyophilized for 24 hours and visualized using a scanning electronmicroscope (Hitachi S-4800 microscope, Schaumburg, Ill.).

Formulation and Characterization of BCMA Peptide-Loaded Liposome-NP.

A thin film hydration method was used to synthesize the BCMApeptide-loaded liposome-NP. The liposomes lipid bilayer was made with amixture of Cholesterol (MW=386.654), DOPC (MW=786.113), and DOTAP(MW=698.542) (Avanti Polar, Alabaster, Ala.). Briefly, a 1 ml stocksolution from the mixture of 3 mM Cholesterol, 5 mM DOPC, and 5 mM DOTAPwas prepared in chloroform. The solvent was evaporated using a rotaryevaporator (RV 10, IKA, Wilmington, N.C.) to yield a thin lipid film atthe base of the flask. The lipid film was subjected to overnight vacuumdrying to remove any residual organic solvent. The next day, hBCMA₇₂₋₈₀peptide dissolved in sodium phosphate (dibasic; pH 11) buffer and 1%DMSO was used to hydrate the lipid film using 10 freeze-thaw cycles(−80° C. and 37° C.), followed by 1 minute of probe sonication on ice toreduce particle sizes to the desired range. The peptide-loadedliposome-NPs were recovered by ultracentrifugation and resuspended inPBS. Blank liposomes were prepared following the same procedure exceptfor the hydration step, where dibasic buffer with 1% DMSO was usedwithout any peptide. Transmission electron microscopy (TEM) was used tocharacterize the surface morphology of the BCMA peptide-loadedliposome-NP. Uranyl acetate (2%) was used as a negative staining tovisualize the BCMA peptide-loaded liposome-NP using TEM (JEM-1000, JEOL,Tokyo, Japan).

BCMA Peptide Encapsulation in PLGA-NP or Liposome-NP.

The level of peptide encapsulation on PLGA-NP or Liposome-NP wasmeasured using the Quantitative Fluorometric Peptide Assay Kit (ThermoFisher) per the manufacturers' suggested protocol. In brief, an equalamount of BCMA peptide in suspension, BCMA peptide loaded in PLGA-NPs(10 μl) or BCMA peptide loaded in liposome-NPs (10 μl) was loaded intriplicate to a 96-well fluorescence-compatible microplate. Blank PLGAor blank liposome were used as the negative control. Next a solution of1:1 Acetonitrile:DMSO (70 μl) and FluoroBrite™ DMEM (20 μl) was added toeach well and incubated for 5 minutes at room temperature in the dark.Acetonitrile:DMSO solution was used as a control to measure thebackground fluorescence. Following incubation, fluorescence was measuredusing a spectrophotometer at Ex/Em at 390 nm/475 nm. The peptideconcentration was determined based on the standard curve (0-1,000 μg/ml)generated in a linear fit.

Generation of Monocyte-Derived Dendritic Cells

Monocyte-derived dendritic cells (DC) were generated substantially asdescribed previously (Bae et al. 2011, Bae et al. 2012). Briefly,monocytes isolated from HLA-A2⁺ normal donors' peripheral bloodmononuclear cells (PBMC) were cultured for 7 days in the presence of1,000 U/ml GM-CSF and 1,000 U/ml IL-4 in RPMI-1640 medium (Gibco-LifeTechnologies) supplemented with 10% FCS. The fresh media containingcytokines is replaced every other day. The immature dendritic cells werecollected from the culture on day 7 for uptake study. Mature DC wereobtained by adding 1,000 U/ml IFN-α plus 10 ng/ml TNF-α, along withfresh GM-CSF and IL-4 in 10% FCS-RPMI, upon immature dendritic cellsgeneration on day 7, and then incubating for an additional three days.Either immature or mature DC were used as antigen-presenting cells(APC).

Binding and Uptake of BCMA Peptide-Encapsulated Nanoparticles byDendritic Cells

Immature human dendritic cells (immDC) were harvested, washed,resuspended in serum-free media (1×10⁶ cells/ml), and aliquoted intowells of a 48-well TC-plate at a final at concentration of 5×10⁵cells/well. Cells were pulsed with 50 ug/ml of BCMA peptide-FITC peptideor BCMA peptide-FITC encapsulated nanoparticles in the presence of 3μg/ml of human β2-microglobulin, and then incubated at 37° C. Peptideloading of immDC was evaluated in a time-dependent manner (0, 30 min, 1hr, 2 hr, 6 hr, 18 hr) by flow cytometry. Additionally, BCMApeptide-FITC uptake was imaged by confocal microscopy (Nikon widefieldMicroscope; Tokyo, Japan) on dendritic cells after a 2 hr peptide pulse.Following incubation, the ImmDC were washed, fixed with 2%paraformaldehyde (Electron Microscopy Sciences, Hatfield, Pa.), andstained with DAPI (Sigma) at 300 nM to identify cell nuclei.

Isolation of Primary CD138+ Tumor Cells from Newly Diagnosed MultipleMyeloma Patients.

Primary CD138⁺ cells were isolated from bone marrow mononuclear cells(BMMC) obtained from both HLA-A2⁺ and HLA-A2⁻ newly diagnosed multiplemyeloma patients using RoboSep® CD138 positive immunomagnetic selectiontechnology (StemCell Technologies), after appropriate informed consent.

Generation of BCMA Peptide-Specific CTL.

BCMA peptide-specific CTL (BCMA-CTL) were generated ex vivo by repeatedstimulation of peripheral blood mononuclear cells isolated from HLA-A2⁺normal donors' with (1) HLA-A2 specific BCMA immunogenic peptide (50ug/ml), (2) a blank nanoparticle (PLGA or liposome) without peptide, or(3) the BCMA peptide (50 ug/ml) encapsulated nanoparticle (PLGA/peptideor liposome/peptide). PBMC were cultured in DMEM medium supplementedwith 10% human AB serum (BioWhittaker) and pulsed weekly with theappropriate BCMA peptide for a total of 4 or 5 cycles. IL-2 (50 U/ml)was added to the T cell cultures two days after the second stimulation.

Phenotypic Characterization of BCMA-CTL Generated by BCMA PeptideEncapsulated Nanoparticle Stimulation.

BCMA peptide-specific CD8⁺ CTL were evaluated for Naïve: Memory celldevelopment and expression of CD28 co-stimulatory molecule by flowcytometry. In addition, BCMA peptide-specific CD8⁺ CTL were assessedwithin the cultures by staining with BCMA peptide-specific Tetramer-PE.Following tetramer staining at 37° C. for 30 min, cells were washed andstained with CD8-FITC and CD28-APC mAbs. After staining, cells werewashed, fixed in 2% paraformaldehyde, acquired using a LSRII Fortessa™flow cytometer, and analyzed using FACS DIVA™ v8.0 (BD) or FlowJov10.0.7 (Tree star, Ashland, Oreg.) software. BCMA-CTL were analyzed forthe presence\frequency of specific memory (central memory, effectormemory)/non-memory populations within the total gated CD3⁺ CD8⁺ T cellpopulation.

Evaluation of Anti-Myeloma Specific Functional Activities of BCMA-CTLGenerated with BCMA Peptide Encapsulated Nanoparticles.

BCMA-specific CTL (N=3) generated with (1) BCMA immunogenic peptide (50μg/ml), (2) blank nanoparticle without peptide, or (3) BCMA peptide (50μg/ml) PGLA/peptide or liposome/peptide nanoparticles, were evaluatedfor their proliferation response and anti-tumor activities against bothHLA-A2⁺ and HLA-A2⁻ myeloma cell lines or primary CD138⁺ tumor cells. Inbrief, BCMA antigen-specific CTL (5×10⁵ cells) proliferation wasmeasured by coculture of CFSE (Molecular Probes) labeled CTL withirradiated (10 Gy) myeloma cells. On day 3, 4, 5 or 6 of CFSE assays,cells were harvested, stained with live/dead-aqua and fluorochromeconjugated anti-CD3 and anti-CD8 mAbs, and analyzed by flow cytometry.BCMA-CTL functional CD107a degranulation (cytotoxicity) and Th1IFN-γ/IL-2/TNF-α cytokine production were analyzed in response tomyeloma cell lines or primary CD138⁺ tumor cells. In brief, therespective BCMA-CTL (5×10⁵ cells) were co-incubated with target cells inthe presence of fluorochrome conjugated anti-CD107a mAb. After 1 hrco-culture, a cocktail of Brefeldin A and Monensin (BD) was added andincubated for an additional 5 hours. The cells were then stained withlive/dead-aqua and fluorochrome conjugated mAbs specific to various cellsurface antigens, fixed and permeabilized, and stained intracellularlywith mAb specific to IFN-γ, IL-2 or TNF-α. Finally, the cells werewashed, fixed in 2% paraformaldehyde, acquired using a LSRII Fortessa™flow cytometer, and analyzed using FACS DIVA™ v8.0 or FlowJo v10.0.7software.

Results

Multiple myeloma (MM) is a B-cell malignancy characterized by the clonalproliferation and accumulation of malignant plasma cells in the bonemarrow, monoclonal protein in the serum and/or urine, and development ofosteolytic bone lesions. Despite recent advances in treatment usingnovel therapeutics, MM remains incurable. Preclinical studies show thatanti-myeloma CD8⁺ CTL can be generated with immunogenic HLA-A2 orHLA-A24 peptides targeting various tumor-associated antigens (TAA)including XBP1, CD138, and CS1. Moreover, vaccination with thesepeptides can generate MM specific immune responses as detected in theclinical trials. To expand the breadth and extent of antigen-specificimmunotherapy and adoptive immunotherapy in myeloma beyond theseantigens, the disclosure provides additional TAA on tumor cells obtainedfrom newly diagnosed MM patients (N=616). Here, the present disclosureprovides a novel heteroclitic peptide specific to BCMA, the receptor forbinding of B cell activating factor (BAFF) and a proliferation-inducingligand (APRIL). Due to its restricted expression pattern on MM cells andplasma cells along with its critical role in promoting MM cell growth,survival and drug resistance, the BCMA antigen is currently beingtargeted with antibodies, immunotoxins, and CAR T; however, thereremains a significant need to design novel delivery systems capable ofinducing more effective myeloma-specific effector cells with a favorabletherapeutic index. To facilitate BCMA peptide delivery into the bonemarrow microenvironment and augment anti-tumor immune responses in thepatients, an approach using nanoparticle which offers potentialprotection of BCMA peptide from enzymatic degradation and overcomes thelimitations of free peptide vaccines by increasing peptide stability,uptake, and delivery was designed. The goal of the nanotechnology-basedcancer vaccine was to elicit more robust BCMA-specific CD8⁺ CTL immuneresponses and anti-tumor activities in MM patients, through longer andbetter T cell stimulation via sustained antigen release and presentationof the immunogenic peptide. Experiments were performed to evaluate twodifferent types of nanoparticles, PLGA and liposome, encapsulating theBCMA peptide. Compared to peptide alone or liposome/peptide, thePLGA/peptide evokes BCMA-specific CD8⁺ T cells with the greatestanti-tumor activities, associated with antigen-specific memory CTLinduction. These results provide the framework for a therapeuticvaccination and/or adoptive immunotherapeutic strategy using PLGAnanovehicle, which has been approved for human use granted by the USFood and Drug Administration (FDA) with biodegradability andbiocompatibility, to efficiently induce BCMA peptide specific anti-tumoractivity and improve patient outcome in myeloma.

Characterization and Quantification of BCMA Peptide-EncapsulatedNanoparticles.

The double emulsion-solvent technique is the most commonly used methodto formulate PLGA NP to stabilize the emulsion. The lyophilizedparticles were resuspended in distilled water for size andzeta-potential measurements using dynamic light scattering. The blankPLGA showed a size of 309.0±4.0 nm (n=3), while peptide loaded PLGA-NPwere smaller in size (257.7±11.5 nm, n=3), which could be attributed tothe interaction between PLGA polymer and the peptide. The zeta-potentialfor both blank and PLGA-NP were −0.06 to −1.1 mV. The polydispersityindex (PDI) of the PLGA-NPs was ≤0.2, indicating a uniform sizedistribution. Blank PLGA or BCMA peptide encapsulated PLGA-NP weresputter coated with gold/palladium and imaged using a scanning electronmicroscope under 20 kV at 50× to further show uniform size distribution(FIG. 32A). In parallel, liposomal formulations were synthesized usinglipid DOTAP to allow interaction with the BCMA peptide. The liposomalloaded BCMA peptide nanoparticles were approximately 172±0.7 d.nm(diameter, nanometers) (n=3) in size and showed a PDI of 0.2, indicatinga uniform size distribution (FIG. 32B). Negative staining with uranylacetate was used to visualize peptide loaded liposomes using TEM, whichalso revealed a uniform size distribution. Peptide loading andencapsulation efficiency for both NP preparations was evaluated usingQuantitative Fluorometric Peptide Assay, based on fluorescence measuredat Ex/Em at 390 nm/475 nm. The peptide encapsulation efficiency (%),which indicates the percentage of peptide loaded in PLGA-NP orliposome-NP over the initial amount of loaded peptide, was 51%±1.15%(n=3) with PLGA or 49%±1.32% (n=3) with liposome (FIG. 32C). Blank PLGAand blank liposome had zero peptide loading as expected. Afterconfirmation\normalization of peptide encapsulation, the PLGA/peptideand liposome/peptide NPs were used as the BCMA antigen source togenerate antigen-specific cytotoxic T lymphocytes (CTL).

Uptake of BCMA Encapsulated Nanoparticles by Dendritic Cells.

BCMA peptide uptake was evaluated using ImmDC generated from monocytesof HLA-A2⁺ donors. Peptide loading efficiency to ImmDC by eachBCMA-nanoparticle type or peptide alone was measured over time by flowcytometry. A higher efficiency of ImmDC peptide loading was detectedwith BCMA encapsulated nanoparticles as compared to peptide alone. Amongthe two nanoparticles evaluated, liposome/peptide displayed faster andmaintained higher levels of peptide loading (100% uptake 30 minutes)over time compared to PLGA/peptide (FIG. 33A). In contrast, PLGA/peptideshowed a gradual increase in peptide loading over time, being detectedat 1 hr (No carrier/Peptide, PLGA/peptide vs. Liposome/Peptide: 10±4%,22±4% vs. 100±0%), increasing at 6 hr (No carrier/Peptide, PLGA/peptidevs. Liposome/Peptide: 42±4%, 59±2% vs. 100±0%), and peaking at 18 hr (Nocarrier/Peptide, PLGA/peptide vs. Liposome/Peptide: 55±8%, 83±5% vs.100±0%) (FIG. 33B). The loading efficiency of PLGA/peptide by ImmDC wasfurther evaluated by confocal microscopy after an 18 hr pulse. HigherImmDC BCMA peptide loading was seen with PLGA/peptide compared topeptide alone (FIG. 33C), confirming that PLGA/peptide formulationenhances BCMA peptide delivery. Further evaluation demonstrated improvedImmDC peptide loading by PLGA/peptide in a time-dependent manner, asmeasured by flow cytometry (No carrier/Peptide vs. PLGA/peptide: 0 hrpulse (baseline)—4% vs. 3%, 2 hr pulse—22% vs. 36%, 4 hr pulse—28% vs.46%, 6 hr pulse—43% vs. 62%) (FIG. 33D). In addition, using T2 cells asantigen-presenting cells, the same pattern of improved APC peptideloading in a time-dependent manner was detected (No carrier/Peptide vs.PLGA/peptide: 30 min pulse—1% vs. 4%, 2 hr pulse—8% vs. 11%, 24 hrpulse—13% vs. 41%) (FIG. 33E). Between the two antigen-presenting celltypes, primary ImmDC displayed a higher efficiency of peptide uptakethan T2 cells. Thus, these results demonstrate the beneficial effect ofboth PGLA/peptide or liposome/peptide to enhance BCMA peptide loading byantigen-presenting cells.

PLGA/Peptide CTL Display the Highest Functional Immune Responses AgainstMultiple Myeloma Cells.

BCMA-specific CTL were evaluated one week after the fourth stimulationfor their tumor-specific activities, following incubation with eitherHLA-A2⁺BCMA⁺ (U266, McCAR) or HLA-A2⁻BCMA⁺ (RPMI) myeloma cells.Representative flow cytometric analyses of BCMA-CTL generated by (1)BCMA peptide itself, (2) PLGA/peptide or (3) liposome/peptidedemonstrated HLA-A2 restricted anti-myeloma activities including CD107adegranulation (FIG. 34A), IFN-γ production (FIG. 34B), IL-2 production(FIG. 34C), and TNF-α production (FIG. 34D), against HLA-A2⁺ U266myeloma cells, but not against HLA-A2⁺ RPMI myeloma cells. Among thenanoparticle generated BCMA-CTL, PLGA/peptide induced superiorantigen-specific CTL, evidenced by their higher level of anti-tumoractivities [CD107a upregulation and IFN-γ/IL-2/TNF-α productions] thanliposome/BCMA peptide-induced CTL. Further analyses confirmed thatPLGA/peptide-CTL generated from additional HLA-A2⁺ donors' (n=3)displayed the highest anti-myeloma activities in response toHLA-A2⁺BCMA⁺ U266 and HLA-A2⁺ BCMA^(+(low)) McCAR, but not to WICmismatched HLA-A2⁻BCMA⁺ RPMI myeloma cells (FIG. 34E). In comparison,liposome/peptide-CTL had a slightly higher level of anti-MM activitiescompared to BCMA peptide-CTL, which were both lower thanPLGA/peptide-CTL. Thus, these results indicate enhanced immunogenicityof BCMA peptide upon PLGA encapsulation, resulting in efficientinduction of poly-functional CTL against multiple myeloma cells in anHLA-A2 restricted manner.

Highest Anti-MM Activities by BCMA-CTL Generated with PLGA EncapsulatedBCMA Peptide Stimulation Against Primary CD138⁺ Tumor Cells from MyelomaPatients.

The myeloma-specific functional activities of BCMA-specific CTL,generated with or without NP encapsulation, were further evaluatedagainst primary CD138⁺ tumor cells from HLA-A2⁺ or HLA-A2⁻ myelomapatients. The three different effector cells generated by stimulationwith BCMA peptide alone, PLGA/BCMA peptide, or liposome/BCMA peptide allshowed minimal background levels pf CD107a degranulation andIFN-γ/IL-2/TNF-α production among the effector cells (FIG. 35A). Amongthe effector CTL, PLGA/peptide-CTL demonstrated the highest anti-MMactivities against primary HLA-A2⁺ CD138⁺ (MM Patient 1) tumor cells[BCMA peptide-CTL vs. PLGA/peptide-CTL vs. Liposome/peptide-CTL: CD107a⁺CTL—15.0% vs. 31.7% vs. 16.2%, IFN-γ⁺ CTL—1.9% vs. 7.4% vs. 3.7%, IL-2⁺CTL—14.3% vs. 32.3% vs. 17.9%, TNF-α⁺ CTL—16.4% vs. 36.8% vs. 19.1%](FIG. 35B). Despite having the highest peptide uptake by DC, the anti-MMactivities of the liposome/peptide-CTL was less than PLGA/peptide-CTLagainst primary HLA-A2⁺ CD138⁺ tumor cells. A similar pattern offunctional anti-MM activities was detected in the effector CTL againstprimary CD138⁺ tumor cells obtained from an HLA-A2⁺ MM patient #2, withthe PLGA/peptide-CTL having the highest activities (FIG. 35C). Incontrast, none of the different BCMA-CTL had anti-tumor activitiesagainst HLA-A2⁺ CD138⁺ tumor cells, thereby demonstratingHLA-A2-restricted immune responses (FIG. 35D). Taken together, theseresults indicate that the highest level of anti-myeloma activities areseen with PLGA/BCMA peptide-CTL against primary CD138⁺ MM cells in anHLA-A2 restricted manner. The results also highlight the potential ofincreased anti-tumor activities through generation of BCMA-specific CTLupon the peptide encapsulation in PLGA.

Increased CD28 Costimulatory Molecule Expression and Peptide-Specific TCells Proliferation by BCMA-CTL Generated with PLGA Encapsulation.

To better understand the mechanism of high anti-tumor activities in theBCMA-specific CTL mediated by PLGA, experiments were performed toevaluate their expression of costimulatory molecule on CD8⁺ T cells andBCMA peptide-specific Tetramer⁺ CTL. Compared to BCMA peptide-CTL,PLGA/peptide CTL demonstrated a unique subset of cells with upregulatedCD28⁺⁺ expression on the CD8⁺ T cells (peptide-CTL vs. PLGA/peptide-CTL:15.6% vs. 32.0%; FIG. 36A upper panel). In addition, PLGA/peptide CTLcontained a higher proportion for BCMA Tetramer⁺ peptide-specific CTL ascompared to BCMA peptide CTL (peptide-CTL vs. PLGA/peptide-CTL: 16.5%vs. 35.0%; FIG. 36A lower panel). In addition, a higher frequency ofbright CD28⁻ cells was detected within the Tetramer-positive as compareto Tetramer-negative CD8⁺ T cells. The PLGA/peptide Tetramer⁺ CTLdisplayed a higher frequency of CD28⁺⁺ bright cells than BCMA peptideTetramer⁺ CTL (51.8% vs. 30.3%). Thus, these results demonstrate thatPLGA/peptide induced CTL have a greater proportion of BCMA-specificTetramer⁺ cells having a unique population of bright CD28⁺⁺BCMA-specific CTL. Next, experiments were performed to demonstrateproliferation of BCMA peptide CTL and PLGA/peptide CTL upon recognitionof their cognate BCMA peptide presented by APC. Both BCMA peptide CTLand PLGA/peptide CTL demonstrated increased proliferation uponrecognition of their cognate BCMA peptide in a time dependent manner(Day 3, Day 4 vs. Day 5: 11%, 18% vs. 33%) as compared to the baselineproliferation of non-BCMA specific CD8⁺ T cells (Day 3, Day 4 vs. Day 5:0%, 1% vs. 4%). Importantly, proliferation occurred earlier inPLGA/peptide-CTL at all time points (Day 3, Day 4 vs. Day 5: 25%, 45%vs. 69%) (FIG. 36B), indicating an increased ability to recognize andrespond to the cognate BCMA peptide. Lastly, Th1 cytokine productiongenerated in response to cognate BCMA peptide. was measured in eacheffector cell population. PLGA/peptide CTL had a higher level of IFN-γproduction (two days incubation—33.8%, four days incubation—60.6%), ascompared to BCMA peptide CTL (two days incubation—31.5%, four daysincubation—42.1%) (FIG. 36C). These results, therefore, demonstrateenhanced peptide-specific CD8⁺ T cell immune responses, proliferation,and IFN-γ production in response to PLGA/peptide-CTL, indicating thatPLGA/peptide encapsulation can increase the immunogenicity of BCMApeptide to generate CTL with higher anti-tumor activities.

Effective Generation of Memory CD8⁺ CTL Associated with EnhancedAnti-Myeloma Activities in Response to Stimulation with PLGA/BCMAPeptide.

Experiments were further performed to characterize and compare memorycell development and the immune functional activities of PLGA/peptide,blank PLGA (control), and peptide alone induced CTL. In CFSE assays,PLGA/peptide CTL displayed higher proliferation in response to HLA-A2⁺U266 cells by (Day 4: 28.90%, Day 6: 44.70%) than BCMA peptide-CTL (Day4: 12.20%, Day 6: 29.20%) at all time points evaluated (FIG. 37A).Effector cells stimulated with PLGA itself as a vehicle displayedminimal proliferation (less than 7%) on 4 days and 6 co-culture. Inaddition, no proliferation was seen in the media controls, providingevidence that CTL proliferation was specific to myeloma cells. Next,experiments were performed to characterize memory cell developmentwithin BCMA peptide CTL and PLGA/peptide CTL. Overall, it was observed agradual increase in CTL memory cell development after each round ofpeptide stimulation: total CD45RO⁺ memory CTL was higher after PLGA/BCMApeptide stimulation (3′ stimulation: 20.7%, 4′ stimulation: 32.8%, 5′stimulation: 93.5%) compared to BCMA peptide (3′ stimulation: 15.3%, 4′stimulation: 19.5%, 5′ stimulation: 79.0%) (FIG. 37B). This pattern ofmemory cell development in the PLGA/peptide CTL and BCMA peptide CTLremained post-2 or post-4 cycles of peptide stimulation (FIG. 37C).Experiments were also performed to characterize specific central andeffector memory subset development in the PLGA/peptide CTL and BCMApeptide CTL. Consistent with total CD45RO⁺ memory development, both thecentral memory (CM) and effector memory (EM) CD8⁺ T cell subsetsgradually increased after 1 cycle of stimulation (PLGA/Peptide—CM 5.3%,EM 8.1%, Peptide—CM 2.9%, EM 5.7%), and further increased after 3 cyclesof stimulation (PLGA/peptide—CM 14.8%, EM 13.2%, Peptide—CM 12.4%, EM11.0%). After the 5-cycle of peptide stimulation, a major difference inthe proportion of central memory and effector memory cell developmentwas observed (PLGA/peptide—CM 42.4%, EM 32.6%, Peptide—CM 3.5%, EM95.4%). In addition, PLGA/peptide CTL maintained a higher proportion ofcentral memory T cells with the highest anti-tumor activities, withoutfurther differentiation to effector memory cells (FIG. 37D).

Maintenance of Central Memory CD8⁺ CTL Associated with EffectiveAnti-Myeloma Activities by PLGA/BCMA Peptide.

The specific anti-MM activities were further investigated within eachmemory CTL subset. Here, it was confirmed the HLA-A2-restrictedanti-myeloma activities of BCMA-specific CTL generated from differentHLA-A2⁺ individuals by stimulation with PLGA/peptide or BCMA peptide.The highest immune functional activities (CD107a upregulation andTh1-type cytokine production) in response to HLA-A2⁺ U266 myeloma cellswere consistently seen in CTL induced by PLGA/peptide (FIGS. 38A, 38B,38C). Importantly, the highest anti-MM activities were found within thecentral memory as compared to the effector memory subsets, as shown byCD107a degranulation (FIGS. 38A, 38C), IFN-γ production (FIGS. 38B,38C), and IL-2/TNF-α production (FIG. 38C). These therefore indicatethat PLGA encapsulated BCMA peptide induces a more robust tumor specificCTL response than BCMA peptide, evidenced by generation and maintenanceof central memory cells within the PLGA/peptide BCMA antigen-specificCTL.

These results support the use of PLGA/BCMA peptide to induce effectiveBCMA CTL with anti-tumor activities in novel vaccination and/or adoptiveimmunotherapy treatment protocols in myeloma.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A peptide comprising: (a) an amino acid sequence that is identical tothe amino acid sequence set forth in any one of SEQ ID NO: 13-17, ordiffers by 1 to 4 amino acid residues, wherein the amino acid atposition 1 of SEQ ID NOs: 13-17 is unaltered; or (b) a first amino acidsequence consisting of an amino acid sequence that is at least 60%identical to any one of SEQ ID NOs: 1-17; and a second amino acidsequence that is heterologous to the first amino acid sequence; whereinthe peptide (i) binds to a major histocompatibility complex (MHC)molecule, or (ii) in association with a MHC molecule, is recognized byan antigen specific T cell receptor on a T cell. 2.-20. (canceled)
 21. Anucleic acid encoding the peptide of claim
 1. 22. A vector comprising anucleic acid encoding the peptide of claim
 1. 23. (canceled)
 24. Acultured cell comprising the vector of claim 22, wherein the nucleicacid is operably linked to a promoter, a regulatory element, or anexpression control sequence. 25.-27. (canceled)
 28. A virus comprising anucleic acid encoding the peptide of claim
 1. 29. (canceled)
 30. Acombination of at least two different peptides, wherein the at least twodifferent peptides are (i) selected from the group consisting ofpeptides having an amino acid sequence set forth in SEQ ID NOs: 13-17;or (ii) selected from the group consisting of peptides having an aminoacid sequence that is at least 60% identical to any one of SEQ ID NOs:13-17, wherein the at least two different peptides (i) bind to a majorhistocompatibility complex (MHC) molecule, or (ii) in association with aMHC molecule, is recognized by an antigen specific T cell receptor on aT cell.
 31. (canceled)
 32. A pharmaceutical composition comprising thecombination of claim 30; and a pharmaceutically acceptable carrier. 33.(canceled)
 34. (canceled)
 35. A composition comprising an isolateddendritic cell, wherein the isolated dendritic cell presents a peptideon its surface, wherein the peptide comprises at least one majorhistocompatibility complex (MHC) class I peptide epitope of one or bothof BCMA antigen (SEQ ID NO: 18) and TACI antigen (SEQ ID NO: 19).36.-89. (canceled)
 90. A method of identifying a T cell antigen receptorsequence for TACI, the method comprising (a) generating and/orproliferating TACI-specific cytotoxic T cells by contacting one or morecytotoxic T cells with one or more antigen presenting cells pulsed witha peptide comprising the amino acid sequence of any one of SEQ ID NOs:15-17 and (b) determining the T cell antigen receptor sequence for TACIin the TACI-specific cytotoxic T cells.
 91. (canceled)
 92. A compositioncomprising: a nanoparticle, and a peptide comprising an amino acidsequence that is at least 60% identical to any one of SEQ ID NOs: 1-17,wherein the peptide (i) binds to a major histocompatibility complex(MHC) molecule, or (ii) in association with a MHC molecule, isrecognized by an antigen specific T cell receptor on a T cell. 93.-105.(canceled)
 106. A method for treating a human subject having a cancer,comprising: administering to the human subject the composition of claim92. 107.-112. (canceled)